Interactive Fly, Drosophila

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 amnX8 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 NF1P2-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 NF1P2. The increased glial thickness of ine1; NF1P2 is fully rescued by heat-shock-induced expression of the NF1+ transgene. However, unlike push, the phenotype of NF1P2 is potentiated only moderately by the eag1 mutation. In contrast, perineurial glial thickness in the push1; NF1P2 double mutant was 2.1 ± 0.15 µm, which is significantly thicker than either push1 or NF1P2, but not significantly different from amnX8. 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; NF1P2 double mutant exhibits a perineurial glial thickness much greater than push1 or NF1P2 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 Galphas. This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galphas, can activate AC. This study also shows that Galphas is the major Galpha isoform in fly brains, and a second AC pathway is defined stimulated by serotonin and histamine requiring NF1 and Galphas. A third, classical Galphas-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/Galphas-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 Galphas; (2) an NF1/Galphas-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 Galphas 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 Galphas (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 Galphas 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 Galphas, 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/Galphas-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).

The GABAA receptor RDL suppresses the conditioned stimulus pathway for olfactory learning: Genetic interactions with NF1

Assigning a gene's function to specific pathways used for classical conditioning, such as conditioned stimulus (CS) and unconditioned stimulus (US) pathway, is important for understanding the fundamental molecular and cellular mechanisms underlying memory formation. Prior studies have shown that the GABA receptor RDL inhibits aversive olfactory learning via its role in the Drosophila mushroom bodies (MBs). This study describes the results of further behavioral tests to further define the pathway involvement of RDL. The expression level of Rdl in the MBs influenced both appetitive and aversive olfactory learning, suggesting that it functions by suppressing a common pathway used for both forms of olfactory learning. Rdl knock down failed to enhance learning in animals carrying mutations in genes of the cAMP signaling pathway, such as rutabaga and NF1, suggesting that RDL works up stream of these functions in CS/US integration. Finally, knocking down Rdl or over expressing the dopamine receptor dDA1 in the MBs enhanced olfactory learning, but no significant additional enhancement was detected with both manipulations. The combined data suggest that RDL suppresses olfactory learning via CS pathway involvement (Liu, 2009b).

The level of Rdl expression in the MBs affects the calcium response observed in these neurons when animals are presented with odor but not shock stimulus. This provided the basis for hypothesizing that RDL might specifically regulate the CS pathway for olfactory learning. Data presented in this study shows that the level of Rdl expression the MBs influences both aversive and appetitive olfactory learning, which share a common CS pathway. Thus, these observations are consistent with the CS pathway-specific hypothesis. Rdl knock down failed to produce enhanced learning when combined with mutations of either the rut or NF1 gene, both of which may be involved in the process of integration of CS and US information. This observation argues against the possibility that RDL acts downstream of CS/US integration, providing further support for RDL's role in the CS pathway (Liu, 2009b).

Prior experiments have shown that blocking neurotransmitter release from dopaminergic neurons impairs aversive olfactory learning but not appetitive olfactory learning, while blocking the synthesis of octopamine impairs appetitive olfactory learning but not aversive olfactory learning. This is consistent with the simple model that the neuromodulators are involved in US pathways for learning, with octopamine delivering only appetitive US (sugar) and dopamine delivering only aversive US (electric shock). This model also suggests that increasing the expression level of dDA1 will increase aversive US input, and thereby enhance aversive learning, as long as other factors such as dopamine release are not limiting. This possibility was tested, and evidence is provided for increased performance with increased expression of dDA1 in the MBs. Since knocking down Rdl increases the CS signal, it follows that combining over-expression of dDA1 with knock down of Rdl might enhance learning synergistically, and produce an even greater enhancement of learning. However, no synergism between these two was detected: although dDA1 over-expression alone and Rdl knock down alone both enhance olfactory learning, the combined treatments failed to produce a significantly higher performance score than either treatment alone. Two possible hypotheses can account for these results. The learning enhancement of either treatment produces performance close to ceiling levels, where no further enhancement can be detected. Alternatively, the dDA1 receptor, and thus the dopamine system, plays some role in the CS pathway that overlaps with RDL, such that the two learning enhancing effects do not sum. The authors prefer the later possibility for two reasons. (1) Functional imaging of the dopaminergic neurons projecting to the MBs using calcium reporters has revealed that these neurons respond not only to shock stimuli presented to the fly, but also to odor stimuli (Riemensperger, 2005). This indicates that the response properties of these neurons are not specific to the US pathway, which is predicted by the 'US pathway only' hypothesis. Rather, dopaminergic neurons respond to the CS and are therefore intertwined in some way with the CS pathway. (2) Flies mutant for the dDA1 gene exhibit impairment in both aversive and appetitive olfactory learning, both of which can be rescued by expressing dDA1 in the MBs (Kim, 2007). This observation suggests that dDA1 may play a role in the CS pathway like RDL. An overriding conclusion is that the model envisioning aversive and appetitive specific US pathway roles for dopamine and octopamine, respectively, is overly simplistic (Liu, 2009b).

The results suggest that the GABAA receptor RDL regulates the CS pathway in Drosophila olfactory learning. The conclusion that the GABAA receptor modulates the CS pathway for learning is not limited to either insects or learning supported by olfactory cues. During taste aversion learning in mice, pre-exposure to the CS of the tastant alone causes latent inhibition where the mice show reduced learning to the CS after pairing the CS with the US. This phenomenon is distinctly absent in male mice carrying a point mutation in the α5 subunit of the GABAA receptor, which is highly expressed in the hippocampus (Gerdjikov, 2008). Since CS information is the only stimulus presented during the pre-exposure period, these results support the role of GABAA receptors in regulating the CS pathway. Extinction is another type of learning where repeated exposure to the CS alone after CS/US conditioning reduces the CR. Systemic administration of a GABAA receptor antagonist blocks the development and expression of extinction in rats during contextual fear learning (Harris, 1998). Since extinction trials are composed of the CS exposure by itself, these results also indicate that GABAA receptors modulate the CS pathway. Moreover, other studies have shown that the surface expression of GABAA receptors increases in the basolateral amygdala after extinction trials following fear conditioning (Chhatwal, 2005). These results indicate that CS exposure alone during extinction is sufficient to modulate the cellular trafficking of GABAA receptors, again indicating a role for GABAA receptors in the CS pathway. The current results, together with these previous studies, strongly indicate that GABAA receptors regulate the CS pathway for associative learning (Liu, 2009b).

A role for GABAA receptors in suppressing learning by regulating the CS pathway has at least two broad implications. (1) It suggests that the receptors provide a gate to the association center (MBs). Other molecules may also provide similar gates, but learning must overcome this negative influence for memory formation to occur. This gate is probably nonspecific relative to odor type, that is, the GABAA receptor gate suppresses learning to most or all odors. It follows that learning must mobilize cellular mechanisms for overriding the gate. These could be at the level of the presynaptic GABAergic neurons, such that the presynaptic neurons release less neurotransmitter after learning, or they could be at the level of the postsynaptic receptor, with receptor expression, sensitivity, or conductance altered by learning. Evidence has been provided for a reduced presynaptic release following learning (Liu, 2009b), but postsynaptic mechanisms may occur as well (Chhatwal, 2005). (2) Events or processes that alter the salience of the CS and its ability to enter into associations might function via altering the presynaptic GABAergic release or the postsynaptic GABAA receptors. For instance, spaced conditioning is generally more effective in producing long-lasting memories compared with massed conditioning. It is possible that the rest period between spaced conditioning trials allows for receptor desensitization, producing a more effective subsequent training trial. Memory acquisition becomes more difficult with age. It could be that aging alters the fluidity of the GABAA receptor gate, making acquisition more difficult (Liu, 2009b).

The receptor tyrosine kinase Alk controls neurofibromin functions in Drosophila growth and learning

Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. This study reports two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1 (Gouzi, 2011).

The prominent expression of Alk in the mammalian and Drosophila CNS and presence of the dAlk ligand Jeb in the embryonic fly CNS, provided the first indication that Alk and Jeb likely participate in the development of the nervous system. Subsequent in vitro studies demonstrated that Alk promoted neuronal differentiation of PC12 or neuroblastoma cell lines, and work in C. elegans implicated its Alk ortholog, scd-2, in the inhibition of presynaptic neuronal differentiation in vivo. Several functions have also been attributed to Drosophila dAlk, including roles in the specification of the intestinal musculature, in retinal axon targeting, and in signaling at the larval neuromuscular junction. The current results establish two novel in vivo functions for the Drosophila dAlk/Jeb receptor/ligand pair, in the regulation of organismal growth and associative learning (Gouzi, 2011).

The results presented in this study lead to a hypothesis that dAlk and dNf1 have opposite roles in controlling neuronal ERK activity during larval development, and therefore determine overall organismal size in a non-cell autonomous manner. In support of this hypothesis, dAlk and dNf1 co-localize extensively in larval neurons, both proteins control ERK activity, and both modulate growth by regulating cell size. In agreement with this conclusion, transgenic neuronal expression of the constitutively active ERK, RlSEM, is sufficient to reduce Drosophila size (Gouzi, 2011).

dAlk is the second active RTK implicated in Drosophila growth control. Previous work demonstrated that the fly homolog of the insulin/IGF1 receptor dInr, regulates both body and organ size. In peripheral tissues, dInr is activated by a family of Insulin-like proteins (dILPs), leading to the activation of the IRS (Chico), PI(3)K (Dp110), PTEN (dPTEN), and Akt/PKB, (dAkt1/dPKB), signaling pathway. Ablating the Insulin Producing Cells (IPCs), or silencing the function of dInr pathway components in the larval CNS resulted in severe growth defects. Notwithstanding the similar growth phenotypes, several lines of evidence argue that dAlk and dInr control growth in fundamentally different ways. Most importantly, organismal growth is impaired when dInr activity or signaling is reduced, whereas a similar phenotype is observed upon dAlk activation. Secondly, dAlk affects organism growth by specifically regulating cell size in a non cell-autonomous manner. In contrast, dInr signaling affects both cell size and number cell-autonomously and non-autonomously. Finally, expression of Jeb or dAlk transgenes in neuroendocrine IPCs using the dILP2-Gal4 driver did not modify pupal size. Thus, although both dAlk and dInr RTKs are involved in body size determination, their mechanisms and sites of action are distinct. This interpretation is consistent with results with the C. elegans Alk homolog Scd-2 shown to function in parallel with or converge with TGF-β signaling, but act independently of the Insulin cascade in dauer determination (Reiner, 2008). However, given that dInr and dAlk are members of the same subfamily of RTKs, a potential explanation for the lack of rescue of dNf1 mutant homozygous larvae with systemic administration of 100 µM of the selective inhibitor TAE684, may be off-target inhibition of dInr at the higher drug concentration (Gouzi, 2011).

Interestingly, S6K (dS6K) resides on a downstream branch of the dInr/PI(3)K signaling pathway and regulates cell size without impinging on cell number. Although the dS6K loss-of-function phenotype resembles the dAlk gain-of-function and dNf1 loss-of-function phenotypes, its mode of action is cell-autonomous. However, it is still tempting to speculate that dAlk and dNf1 ultimately affect neuroendocrine signals that affect dS6K activity in peripheral tissues (Gouzi, 2011).

Increasing signaling through the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway has been reported to suppress the size defect of dNf1 mutants. This among other findings, have led some to propose that dNf1 regulates Ras activity and cAMP levels independently. In contrast, an investigation of the cAMP/PKA sensitive dNf1 mutant growth defect argued that aberrantly upregulated Ras/ERK signaling in Ras2-expressing larval neurons was its proximal cause. The current results further support the latter explanation implicating a Ras/ERK signaling defect downstream of dAlk as the cause of size defects in dNf1 mutants. Then, how could elevated cAMP/PKA signaling rescue decreased body size? Because neuroendocrine signals can activate the cAMP pathway, it is possible that defective dAlk/Ras/ERK signaling in dNf1 mutants may lead to a neuroendocrine deficiency, which is restored by increasing cAMP/PKA signals (Gouzi, 2011).

In Drosophila, the dAlk/Jeb receptor-ligand pair has been shown to act in an antrerograde signaling pathway essential for assembly of the neuronal circuitry of the fly visual system. However, loss of either Alk or Jeb did not appear to impair assembly of functional synapses with normal postsynaptic responses at the larval neuromuscular junction, indicating that they do not participate in CNS development. In agreement, pan-neuronal, spatially restricted attenuation or unregulated activation of Alk throughout development did not appear to yield gross structural defects in the adult brain or alter naïve behavioral responses to the stimuli used for conditioning. Hence, it is unlikely that the learning phenotypes described in this study are the result of developmental alterations in the CNS. In fact, dAlk seems to be acutely required for normal learning as limiting modulation of its activity to the adult CNS results in phenotypes on its own and also modifies the learning deficits of dNf1 mutants. Moreover, the function of dAlk and dNf1 in associative learning is independent of body size as the learning reverted to normal by dAlk abrogation in the small-sized dNf1 mutant homozygotes. Interestingly, the C. elegans Jeb homolog Hen-1 is required non-cell autonomously in the mature nervous system for sensory integration and associative learning (Ishihara, 2002). Collectively then, these studies in C. elegans, mice and the current data strongly support an evolutionary conserved role for Alk signaling in adult associative learning and memory (Gouzi, 2011).

Elevated dAlk/Jeb signaling outside the MBs impaired olfactory learning, while its abrogation increased learning efficiency. These are results are consistent with the enhanced performance in a hippocampus-dependent task described for Alk knockout mice (Bilsland, 2008). It is proposed then, that Alk signaling normally functions to limit the strength of the CS/US associations, in effect providing a putative threshold required to be overcome for specific and efficient association of the stimuli. A GABAergic neuron outside the MBs, the Anterior Paired Lateral (APL), was recently reported to similarly suppress olfactory learning and its silencing yielded enhanced performance. Interestingly, a decrease in presynaptic GABA release or abrogation of the GABAA receptor, RDL in the post-synaptic mushroom body neurons resulted in enhanced learning. Whether Ras2-Gal4 is expressed in the APL neuron and dAlk also functions in this neuron to suppress learning are questions currently under investigation (Gouzi, 2011).

Interestingly, a recent study suggested that the learning defects of Nf1+/− mice are attributed to increased ERK-mediated phosphorylation of synapsin I in hippocampal inhibitory neurons and concomitant increase in GABA release. In accord, a GABAA receptor antagonist enhanced learning in Nf1+/− mice and controls, and reversed LTP defects in the mutants. Similarly, elimination of the dAlk-mediating inhibition in Drosophila Ras2-expressing neurons enhanced learning, potentially via attenuation of ERK phosphorylation. In support of this notion, this study shows that constitutive activation of ERK in adult Ras2-expressing neurons precipitates learning deficits. Collectively, these results together with the reported learning deficits of Drosophila synapsin mutants, suggest that a mechanism similar to that proposed for vertebrates may also regulate Nf1-dependent learning in flies (Gouzi, 2011).

In mice, a decrease in Nf1 levels in heterozygous mutants increased Ras/ERK signaling and precipitated Long-Term Potentiation (LTP) and spatial learning deficits. These deficits were reversed upon genetic or pharmacological inhibition of Ras signaling. The current results demonstrate that dAlk inhibition reversed the impaired learning of dNf1 mutants and since this is the first 'kinase-active' RTK shown to be involved in this process in flies, it provides independent support for Ras/ERK hyperactivation as causal of these learning defects. Then, how can the reported phenotypic reversal of Nf1 learning deficits by expressing the PKA catalytic subunit throughout the fly be explained? It is hypothesized that the MBs are functionally downstream of the dAlk/dNf1 neurons and elevated PKA activity within the former could result in normal learning. Future work will focus on addressing the merits of these hypotheses regarding the mechanisms underlying the size and learning defects of dNf1 mutants (Gouzi, 2011).

A recent report Buchanan (2010) suggested that dNf1 mRNA is found in the mushroom bodies and in agreement, immunohistochemical results of the current study demonstrate that dNf1 is present within the mushroom body calyces. Protein synthesis-dependent memory defects in Nf1 mutants were rescued upon MB-limited expression of the same full-length transgene as was used in this study. Since memory deficits were not examined in the current work, this complements the current data and suggests a function for dNf1 within the MBs. In contrast, the current data indicate that dNf1 expression in the MBs is not sufficient for learning/3 min memory. Three common MB drivers including the most specific MB247 and the most broadly expressed OK107-Gal4, did not rescue learning in Nf1 mutants by expressing dNf1. It is suggested therefore that rescue described by Buchanan and Davis was mediated largely by c739-Gal4 transgene expression in neurons extrinsic to MBs where Elav, Ras2 and Alk(38)-Gal4 are expressed, perhaps in combination with expression within MB-intrinsic neurons. The neuronal circuits where dNf1 and dAlk are required for normal learning are the subject of ongoing investigations (Gouzi, 2011).

This study identifies dAlk as the first RTK to functionally interact with Nf1 in Drosophila, raising the important question whether a similar functional relationship exists in mammals. Suggestive evidence argues that this may indeed be the case. Thus, Alk and NF1 extensively colocalize in the mammalian CNS during the same developmental periods. Additionally, excess Alk expression or activation has been reported in astrocytomas, gliomas, neuroblastomas and pheochromocytomas, in which loss of NF1 expression has also been found. Based on identification of Alk as a bona-fide RTK that initiates a Ras/ERK cascade regulated by Nf1, this suggests that Alk inhibition may rescue not only the phenotypes reported in this study, but also other symptoms that have been previously associated with Nf1 loss and ERK over-activation. It was recently reported that knockdown of NF1 expression renders a neuroblastoma cell line resistant to retinoic acid-induced differentiation, and that NF1 deficient neuroblastoma tumors have a poor outcome. The current results suggest that Alk inhibition may provide an intervention strategy in such cases. Finally, the findings reported in this study, combined with the lack of overt abnormalities in Alk knock-out mice, provide a rationale for further explorations of Alk as a potential therapeutic target in NF1 (Gouzi, 2011).

A distinct set of Drosophila brain neurons required for neurofibromatosis type 1-dependent learning and memory

Nonspecific cognitive impairments are one of the many manifestations of neurofibromatosis type 1 (NF1). A learning phenotype is also present in Drosophila melanogaster that lack a functional neurofibromin gene (nf1). Multiple studies have indicated that Nf1-dependent learning in Drosophila involves the cAMP pathway, including the demonstration of a genetic interaction between Nf1 and the rutabaga-encoded adenylyl cyclase (Rut-AC). Olfactory classical conditioning experiments have previously demonstrated a requirement for Rut-AC activity and downstream cAMP pathway signaling in neurons of the mushroom bodies. However, Nf1 expression in adult mushroom body neurons has not been observed. This study addresses this discrepancy by demonstrating (1) that Rut-AC is required for the acquisition and stability of olfactory memories, whereas Nf1 is only required for acquisition, (2) that expression of nf1 RNA can be detected in the cell bodies of mushroom body neurons, and (3) that expression of an nf1 transgene only in the alpha/beta subset of mushroom body neurons is sufficient to restore both protein synthesis-independent and protein synthesis-dependent memory. These observations indicate that memory-related functions of Rut-AC are both Nf1-dependent and -independent, that Nf1 mediates the formation of two distinct memory components within a single neuron population, and that understanding of Nf1 function in memory processes may be dissected from its role in other brain functions by specifically studying the alpha/beta mushroom body neurons (Buchanan, 2010).

Neurofibromatosis Type 1 (NF1) is an autosomal, dominant genetic disorder that afflicts approximately one in every 3500 individuals. Like other clinical manifestations of NF1, expression and penetrance of cognitive phenotypes varies and may include deficiencies of visual-spatial processing, executive function, and attention. Homologs of human Nf1 in mouse and Drosophila melanogaster share significant identity at the protein level, and animal models in both species were developed shortly after the human Nf1 gene was cloned. Both models demonstrate cognitive phenotypes, and insights gained through animal studies have shed light on the genetic and biochemical basis of these defects (Buchanan, 2010).

Drosophila has been utilized extensively for expanding basic understanding of memory, making it ideal for investigating NF1 cognitive deficits. After olfactory classical conditioning, Drosophila form protein synthesis-independent early memories (PSI-EM), comprised of short-term memory (STM) tested at 3 minutes after training, middle-term memory (MTM) often tested at 3 hours after training, and protein synthesis-dependent long-term memory (PSD-LTM), tested at 24 hours after conditioning. The nf1 mutant flies demonstrate deficiencies in PSI-EM and PSD-LTM. A current model postulates that Nf1 contributes to PSI-EM through stimulation of the rutabaga-encoded adenylyl cyclase (Rut-AC). Stimulation of Gαs-dependent AC activity requires only the Nf1 C-terminal domain. The PSI-EM phenotypes of nf1, rut-AC, and nf1/rut-AC mutants are similar, both genes are required at the time of learning, and either ubiquitous expression of a constitutively active protein kinase A (hsPKA*) transgene or neuronal expression of a Nf1 C-terminal domain transgene rescues the nf1 phenotype. Furthermore, the current model also postulates that Nf1 contributes to PSD-LTM through regulation of Ras via its GAP-related domain (GRD). Stimulation of Ras-dependent AC activity is absent in nf1 mutants, but transgenic expression of the Nf1-GRD restores this activity and improves the PSD-LTM phenotype of nf1 mutant (Buchanan, 2010).

It is surprising that endogenous Nf1 expression has not been observed in adult mushroom body (MB) neurons. MB neurons are essential for olfactory memory formation, and Rut-AC is preferentially expressed in these neurons. Rescue experiments demonstrated that transgenic expression of rut-AC in α/β and γ MB neurons restores normal memory in homozygous mutants. If Nf1 indeed stimulates Rut-AC activity during learning, it is probably expressed, and required, in MB neurons. This study explores whether Nf1 and Rut-AC are involved in the same operational phase of learning, whether they are expressed in the same neurons, whether both are required in the same neurons for rescue of PSI-EM, and whether the Ras-mediated function of Nf1 is required in overlapping neurons. A role is reported for Rut-AC in memory stability that is Nf1-independent, nf1 expression in MB neurons was observed, and a requirement for nf1 expression in α/β MB neurons was observed for both PSI-EM and PSD-LTM (Buchanan, 2010).

Regardless of species being studied, neurofibromin is involved in many different brain activities including, but not limited to, cognitive processes, circadian rhythms, cortical development, and glial development. Even within the cognitive realm, Nf1 function depends on the context of specific training conditions. Protein synthesis-independent short- and middle-term memories appear to require an activation of Rut-AC by Nf1, whereas protein synthesis-dependent long-term memory requires an additional modulation of Ras activity. By rescuing the performance of homozygous mutants, this study has demonstrated that expression of Nf1 in adult α/β mushroom body neurons is sufficient to support all forms of Nf1-dependent memory. A requirement was revealed for Nf1 during acquisition. Together, thee observations expand current understanding of Nf1 and Rut-AC functions and challenge current models of mushroom body neuron activity in olfactory memory formation (Buchanan, 2010).

Rut-AC is required in both α/β and γ mushroom body neurons for complete rescue of rut STM deficits (Akalal, 2006), yet this study has shown that Nf1 is required only in the α/β mushroom body neurons. It is unclear why Rut-AC activation would only require Nf1 in one subset of neurons. One possibility is that the Nf1 stimulation of Rut-AC in α/β neurons during acquisition may indirectly facilitate, through unknown signals, Rut-AC activity in the γ neurons. Prior results have been interpreted to suggest that there are communication loops that exist between certain types of mushroom body neurons, and with extrinsic mushroom body neurons for normal learning and consolidation. A similar process could allow Rut-AC activation in γ neurons to be indirectly dependent upon Nf1 in α/β mushroom body neurons. Alternatively, it could be that the Rut-AC is dependent upon Nf1 in the α/β mushroom body neurons for its role in learning but Nf1-independent in γ mushroom body neurons (Buchanan, 2010).

For rescue of protein synthesis-dependent long-term memory (PSD-LTM), both Rut-AC and Nf1 expression are required only in α/β neurons, suggesting that their interaction is necessary to support this form of memory as well. A recent study concluded that the Nf1-GAP-related domain, which has been shown to mediate an adenylyl cyclase activity (Hannan, 2006), is necessary and sufficient for Nf1-dependent LTM (Ho, 2007). In contrast to Rut-AC, this adenylyl cyclase activity is stimulated by Ras and is Gαs-independent. It is important to note, however, that the Nf1-GRD domain only partially rescued the LTM phenotype of nf1 mutants. Full rescue of LTM required a full-length nf1 transgene. Together, these data and and the current study suggest that Nf1 simultaneously mediates the activation of both AC signaling pathways in α/β neurons to facilitate new protein synthesis and the formation of long-lasting memory (Buchanan, 2010).

Early work on the role of Rut-AC in olfactory associative memory suggested that this adenylyl cyclase plays a role in behavioral acquisition. Using an olfactory avoidance assay, it was suggested that rutabaga mutants could obtain normal performance with more intense training. A delay was observed in the acquisition of olfactory memory in rutabaga mutants, which require 3 times the amount of training as controls to overcome. A similar delay in acquisition was discovered for nf1 mutants, consistent with the hypothesis that Nf1 is required for G-protein activation of Rut-AC during learning. The results also demonstrate that Rut-AC is essential for the stability of olfactory memory. However, this function is independent of an interaction with Nf1. It is believed that the association of Nf1 and Rut-AC may be transient, only required for the initial activation of Rut-AC in its role as a molecular coincidence detector in α/β neurons (Tomchik, 2009). If this model were true, memory stability would therefore require continued stimulation of Rut-AC molecules via an independent and perhaps spatially distinct mechanism that does not require Nf1 (Buchanan, 2010).

Recent efforts have attempted to assign temporal and operational phases of olfactory memory processing to distinct regions within the adult olfactory system. Upon pairing of odor and electric shock, new projection neuron synapses are recruited to the odor representatio. Pairing dopamine application with neuronal depolarization in adult brain preparations results in a Rut-AC-dependent synergistic increase of cAMP in both α and α’ lobes (Tomchik, 2009), and memory acquisition requires synaptic transmission from α’/β’ neurons. Although it was demonstrated that both Nf1 and Rut-AC are required for memory acquisition, neither of these need be expressed in α'/β' neurons. It is therefore proposed that memory acquisition cannot be thought of as a specific event involving a distinct neuronal subset. Rather, a model is envisioned in which the pairing of odor and electric shock induces a change on the neuronal systems level. Each individual neuron subset may register this change in a different way, but every change is in some way necessary for memory acquisition as a whole. Additional work will be required to determine whether memory consolidation, retrieval, or processing of longer-term memories also require plasticity throughout the entire olfactory system (Buchanan, 2010).

It is clear from the data herein that Nf1 function is required in the adult brain, in α/β neurons defined by the c739-gal4 driver, for PSI-EM formation and for PSD-LTM formation. By identifying a minimal region in which Nf1 expression is required, it is now possible to isolate its role in memory formation from others that may occur in the brain. This mapping promises a more accurate analysis of Nf1-dependent memory and insights into both memory processing as a whole and into the cognitive deficits associated with Neurofibromatosis Type 1 (Buchanan, 2010).

Gamma neurons mediate dopaminergic input during aversive olfactory memory formation in Drosophila

Mushroom body (MB)-dependent olfactory learning in Drosophila provides a powerful model to investigate memory mechanisms. MBs integrate olfactory conditioned stimulus (CS) inputs with neuromodulatory reinforcement (unconditioned stimuli, US), which for aversive learning is thought to rely on dopaminergic (DA) signaling to DopR, a D1-like dopamine receptor expressed in MBs. A wealth of evidence suggests the conclusion that parallel and independent signaling occurs downstream of DopR within two MB neuron cell types, with each supporting half of memory performance. For instance, expression of the Rutabaga (Rut) adenylyl cyclase in γ neurons is sufficient to restore normal learning to rut mutants, whereas expression of Neurofibromatosis 1 (NF1) in α/β neurons is sufficient to rescue NF1 mutants. DopR mutations are the only case where memory performance is fully eliminated, consistent with the hypothesis that DopR receives the US inputs for both γ and α/β lobe traces. This study demonstrates, however, that DopR expression in γ neurons is sufficient to fully support short- and long-term memory. It is argued that DA-mediated CS-US association is formed in γ neurons followed by communication between γ and α/β neurons to drive consolidation (Qin, 2012).

Because DopR is thought to mediate the US information, identification of the spatial requirements of this receptor pinpoints the initial site of CS-US coincidence detection. To date, most genetic and circuit manipulations suggest that olfactory memory performance at a given retention interval can be dissected into distinct and independently disruptable mechanisms acting in parallel in distinct neuronal cell types. For example, the STM defects of rut and NF1 can be rescued with expression in γ for rut and β/γ neurons for NF1. Experimental dissections of the circuits required for LTM have suggested a major role for β/γ neurons as well as for ellipsoid body (eb) and DAL neurons. Such findings have been interpreted as supporting the idea of independent signaling for parallel memory traces as well as sequential action in different cell types to support a single memory mechanism. The current findings demonstrate that DopR expression in MBs is sufficient to support both rut-dependent and rut-independent forms of CS-US association leading to STM, as well as to consolidated ARM and LTM. This conclusion also generalizes to three different combinations among five different odors, providing strong evidence that the functional distinctions between KC classes are not artifacts caused by differences in the population of neurons involved in coding each odor percept. With each of these odor combinations and memory phases, there also was no case where expression in α/β or α'/β' populations was sufficient or necessary to provide substantial rescue of dumb2 (a piggyBac insertion in the first intron of the DopR locus) mutants (Qin, 2012).

Together, this set of findings pinpoints the DopR-mediated inputs for STM, MTM, ARM, and LTM to the γ neuron population of MB KCs. This conclusion is consistent with findings from previous attempts to map the subset of DA neurons that convey the US to MBs using either inhibition or activation of neural transmission to block or mimic the US signal. In these studies, the largest magnitude effects were seen with stimulation of MB-MP1, a neuron in the PPL1 cluster of DA neurons (although it should be noted that smaller magnitude effects also were seen for several other DA cell types), which is sufficient to substitute for the US. Although inhibition of MB-MP1 neurons has not been demonstrated to block learning, these DA neurons likely participate in mediating at least a portion of the US stimulus for aversive conditioning. MB-MP1 neurons project to the base of the peduncle, occupied by the axons of α/β neurons and the heel of the MB, which is comprised largely of γ neurons. As an independent validation of the hypothesis that these MB-MP1 neurons provide direct input to γ neurons, the GFP reconstituted across the synapse (GRASP) method was used to visualize putative synaptic connections in the heel between these two cell types (Qin, 2012).

The fact that γ lobe expression of DopR is sufficient to restore not only STM but also both ARM and LTM is noteworthy. Previous attempts to map the neural circuits for olfactory memory have revealed roles for α/β lobes in particular for consolidated memory. Because massed and spaced training experiments consist of repetitive training rather than the single training trial used for STM and MTM, differences in circuit requirements could in principle derive from training paradigm-dependent differences in the CS-US association circuit, as appears to be true for appetitive reinforcement. But this appears not to be the case for DopR function in aversive reinforcement, because full rescue of these consolidated forms of memory were obtained with γ lobe expression of DopR (Qin, 2012).

How can this conclusion be reconciled with the requirement for downstream signaling molecules within α/β lobe neuron, as well as in downstream eb neurons and dorsal-anterior-lateral (DAL) neurons? Three possible explanations are seen, that are not mutually exclusive. First, it is possible that US information is deconstructed into more than one pathway, mediated by different receptors. These could include additional DA receptors, or other neurotransmitter systems such as serotonin. It is worth noting that DA inputs to MBs also have been implicated in hunger/satiety modulation of appetitive memory retrieval, and DopR signaling also has been implicated in several forms of arousal that in principle could represent a component of the reinforcement signal that could be separate from a more specific perceptual representation of the shock experience. The findings nevertheless lead to the conclusion that any additional US information depends critically on DopR-mediated DA signaling in the γ lobe population of neurons. A second possibility worth considering stems from the finding that output from α/β lobe, eb, and DAL neurons are each required for retrieval depending on the retention interval measured. Thus a model cannot be formally ruled out in which all of the functional impacts of various manipulations of α/β lobe derive from defects in retrieval. This would be difficult to fathom for cases such as NF1 rescue of STM and Rut function for LTM, but in principle this interpretation is possible. The third possibility is that consolidation of the γ lobe CS-US association involves signaling within α/β lobe neurons, as well as in downstream eb neurons and DAL neurons. Such a model predicts communication between the γ lobe and the rest of MBs during training and/or afterward (Qin, 2012).

Genetic and functional studies implicate synaptic overgrowth and ring gland cAMP/PKA signaling defects in the Drosophila melanogaster neurofibromatosis-1 growth deficiency

Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. Among Drosophila Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3'-5' cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue 486 1st and 2nd chromosome deficiencies that uncover >80% of annotated genes were screened for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, it was shown that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting the contention that ALK might be a therapeutic target in NF1, this study reports that human ALK is expressed in cells that give rise to NF1 tumors and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man (Walker, 2013).

Anaplastic lymphoma kinase acts in the Drosophila mushroom body to negatively regulate sleep

Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. This study shows that Alk mutants have increased sleep. Using a targeted RNAi screen the negative effects of Alk on sleep was localized to the mushroom body, a structure important for both sleep and memory. Mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes (Bai, 2015).

Though a few studies implicate Alk orthologs in regulating behaviors such as decision-making, cognition, associative learning and addiction, most functional studies demonstrate various developmental roles for Alk. This study acutely induce a long-sleep phenotype by taking advantage of a temperature-sensitive allele, Alkts, revealing that Alk regulates sleep directly rather than through developmental processes. Mutations in Nf1, a gene encoding a GAP that regulates the Ras/ERK pathway activated by ALK, also cause a sexually dimorphic short-sleep phenotype. Thus this study establishes a novel in vivo function for both Alk and Nf1 and shows they interact with each other to regulate sleep (Bai, 2015).

Many downstream signaling pathways have been proposed for ALK, among them Ras/ERK, JAK/STAT, PI3K and PLCγ signaling. ERK activation through another tyrosine receptor kinase Epidermal growth factor receptor (EGFR) has been linked to increased sleep, while this study shows that Alk, a positive regulator of ERK, inhibits sleep. It is noted that ERK is a common signaling pathway targeted by many factors, and may have circuit- specific effects, with different effects on sleep in different brain regions. Indeed, neural populations that mediate effects of ERK on sleep have not been identified. The dose of ALK required for ERK activation might also differ in different circuits. Region-specific effects of Alk are supported by a GAL4 screen, in which down-regulation of Alk in some brain regions even decreased sleep. The overall effect, however, is to increase sleep, evident from the pan-neuronal knockdown. It was found that the mushroom body, a site previously implicated in sleep regulation and learning, requires Alk to inhibit sleep. Interestingly, the expression patterns of Alk and Nf1 overlap extensively in the mushroom body, suggesting that they may interact here to regulate both sleep and learning. However, it was previously shown that Alk activation in the mushroom body has no effect on learning. The mushroom body expression in that study was defined with MB247 and c772, both of which also had no effects on sleep when driving Alk RNAi. The spatial requirement for Nf1 in the context of learning has been disputed in previous studies with results both for and against a function in the mushroom body. The discrepancies between these studies could result from: 1) varied expression of different drivers within lobes of the mushroom body, with some not even specific to the mushroom body; 2) variability in the effectiveness and specificity of MB-Gal80 in combination with different GAL4s. It was confirmed that the MB-Gal80 manipulation eliminated all mushroom body expression and preserved most if not all other cells with 30Y, 386Y and c309. Future work will further define the cell populations in which Alk and Nf1 interact to affect sleep (Bai, 2015).

A substantial sleep decrease was observed in Nf1 male flies compared to control flies. However, sleep phenotypes in Nf1 female flies are inconsistent. It is unlikely that unknown mutations on the X chromosome cause the short-sleeping phenotype because 7 generation outcrosses into the control iso31 background started with swapping X chromosomes in Nf1P1 and Nf1P2 male flies with those of iso31 flies. In support of a function in sleep regulation, restoring Nf1 expression in neurons of Nf1 mutants reverses the short sleep phenotype to long sleep in both males and females. This does not result from ectopic expression of the transgene as expressing the same UAS-Nf1 transgene in wild-type flies has no effect. It was hypothesized that Nf1 promotes sleep in some brain regions and inhibits it in others, and sub-threshold levels of Nf1, driven by the transgene in the mutant background, tilt the balance towards more sleep. As reported in this study, Alk also has differential effects on sleep in different brain regions, as does protein kinase A, thus such effects are not unprecedented. Severe sleep fragmentation was also observed in Nf1 mutants, which suggests that they have trouble maintaining sleep (Bai, 2015).

The sex-specific phenotypes of Nf1 mutants may reflect sexually dimorphic regulation of sleep. A recently published genome-wide association study of sleep in Drosophila reported that an overwhelming majority of single nucleotide polymorphisms (SNPs) exhibit some degree of sexual dimorphism: the effects of ~80% SNPs on sleep are not equal in the two sexes. Interestingly, sex was found to be a major determinant of neuronal dysfunction in human NF1 patients and Nf1 knock-out mice, resulting in differential vision loss and learning deficits. The sex-dimorphic sleep phenotype in Nf1 flies provides another model to study sex-dimorphic circuits involving Nf1. Interestingly, a prevalence of sleep disturbances have recently been reported in NF1 patient, suggesting that NF1 possibly play a conserved function in sleep regulation (Bai, 2015).

An attractive hypothesis for a function of sleep is that plastic processes during wake lead to a net increase in synaptic strength and sleep is necessary for synaptic renormalization. There is structural evidence in Drosophila to support this synaptic homeostasis hypothesis (SHY): synapse size and number increase during wake and after sleep deprivation, and decrease after sleep. However, little is known about the molecular mechanisms by which waking experience induces changes in plasticity and sleep. FMRP, the protein encoded by the Drosophila homolog of human fragile X mental retardation gene FMR1, mediates some of the effects of sleep/wake on synapses. Loss of Fmr1 is associated with synaptic overgrowth and strengthened neurotransmission and long sleep. Overexpressing Fmr1 results in dendritic and axonal underbranching and short sleep. More importantly, overexpression of Fmr1 in specific circuits eliminates the wake-induced increases in synapse number and branching in these circuits. Thus, up-regulation of FMR accomplishes a function normally associated with sleep (Bai, 2015).

It is hypothesized that Alk and Nf1 similarly play roles in synaptic homeostasis. They are attractive candidates for bridging sleep and plastic processes, because: 1) Alk is expressed extensively in the developing and adult CNS synapses. In particular, both Alk and Nf1 are strongly expressed in the mushroom body, a major site of plasticity in the fly brain. 2) Functionally, postsynaptic hyperactivation of Alk negatively regulates NMJ size and elaboration. In contrast, Nf1 is required presynaptically at the NMJ to suppress synapse branching. 3) Alk and Nf1 affect learning in adults and they functionally interact with each other in this process. It is tempting to speculate that in Alk mutants, sleep is increased to prune the excess synaptic growth predicted to occur in these mutants. Such a role for sleep is consistent with the SHY hypothesis. The SHY model would predict that Alk flies have higher sleep need, which is expected to enhance rebound after sleep deprivation. While the data show equivalent quantity of rebound in Alk mutants, this study found that they fall asleep faster than control flies the morning after sleep deprivation, suggesting that they have higher sleep drive. Increased sleep need following deprivation could also be reflected in greater cognitive decline, but this has not yet been tested for Alk mutants. It is noted that Nf1 mutants have reduced sleep although their NMJ phenotypes also consist of overbranched synapses. It is postulated that their sleep need is not met and thus results in learning deficits. Clearly, more work is needed to test these hypotheses concerning the roles of Alk and Nf1 in sleep, learning, and memory circuits (Bai, 2015).


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Neurofibromin 1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation

date revised: 15 March 2014

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