paper type

aDDeNDUM

rare Diseases 2, e28341; 2014; © 2014 Landes Bioscience

A Drosophila screen identifies neurofibromatosis-1 genetic modifiers involved in systemic and synaptic growth
James A Walker1,2,* and André Bernards1,2,*
Massachusetts General Hospital Center for Cancer Research and Harvard Medical School; Charlestown, MA USA; 2Center for Human Genetic Research; Massachusetts General Hospital; Boston, MA USA
1

Keywords: cAMP/PKA signaling, Drosophila melanogaster, genetic model, neurofibromatosis, Ras signaling, synaptic morphology Abbreviations: NF1, Neurofibromatosis type 1; GAP, GTPase activating protein; ALK, anaplastic lymphoma kinase; CNS, central nervous system; NMJ, neuromuscular junction

N

*Correspondence to: James A Walker; Email: jwalker@helix.mgh.harvard.edu; André Bernards; Email: abernards@helix.mgh.harvard.edu Submitted: 01/06/2014; Revised: 02/12/2014; Accepted: 02/24/2014; Published Online: 02/25/2014 http://dx.doi.org/10.4161/rdis.28341 Addendum to: Walker JA, Gouzi JY, Long JB, Huang S, Maher RC, Xia H, Khalil K, Ray A, Van Vactor D, Bernards R, et al. Genetic and functional studies implicate synaptic overgrowth and ring gland cAMP/PKA signaling defects in the Drosophila melanogaster neurofibromatosis-1 growth deficiency. PLoS Genet 2013; 9:e1003958; PMID:24278035; http://dx.doi.org/10.1371/journal. pgen.1003958

eurofibromatosis type 1 (NF1) is caused by loss of a negative regulator of Ras oncoproteins. Unknown genetic modifiers have been implicated in NF1’s characteristic variability. Drosophila melanogaster dNf1 phenotypes include cognitive deficits and reduced growth, both of which resemble human symptoms. We recently reported results of a screen for dominant modifiers of dNf1 growth. Suppressors include the dAlk tyrosine kinase and its activating ligand, two other genes involved in Ras/ERK signal transduction, the synaptic scaffold Dap160 and the CCKLR-17D1 drosulfakinin receptor. Additional modifiers include several genes involved in cAMP/PKA signaling. Providing mechanistic insights, dAlk, jeb, and CCKLR-17D1 also suppress a dNf1 synaptic overgrowth defect, and increasing cAMP/PKA signaling in the neuroendocrine ring gland rescued the dNf1 growth deficiency. Finally, among the several suppressors identified in our screen, we specifically implicate ALK as a potential therapeutic target by showing that NF1-regulated ALK/RAS/ERK signaling is conserved in human cells. RASopathies are a group of clinically related genetic disorders caused by defects in RAS/ERK signal transduction.1 NF1 is among the most common members of this group, affecting an estimated 1 in 3000 individuals in all ethnic groups. High degrees of variability and unpredictability are among the hallmarks of NF1. Patients

are predisposed to developing a variety of symptoms, the most common of which include benign but potentially highly disfiguring peripheral nerve associated tumors, termed neurofibromas. Malignant tumors, including peripheral nerve sheath tumors, are also strongly associated with NF1. Frequent non-tumor symptoms include skeletal and skin pigmentation abnormalities, reduced overall growth, and cognitive deficits, the latter seen in 50–70% of children with NF1.2 NF1 is caused by mutations that impact the function(s) of neurofibromin, a large and evolutionarily conserved GTPase Activating Protein (GAP) for Ras oncoproteins.3 Neurofibromin and other RasGAPs accelerate the conversion of active Ras-GTP into inactive RasGDP by stimulating the low intrinsic rate of Ras-GTP hydrolysis. While excessive Ras signaling upon loss of neurofibromin is undoubtedly a major cause of NF1 defects, evidence has also been presented that neurofibromin, in Ras-dependent or Ras-independent ways, acts as a positive mediator of adenylyl cyclase activity.4-6 To shed light on the functions of neurofibromin, the molecular pathways involved in NF1 defects and the identity of modifier genes implicated in the characteristic variability of this disease,7 we previously generated loss-of-function mutants of a highly conserved Drosophila melanogaster dNf1 ortholog. Homozygous dNf1 null mutants are viable and fertile, but show a 15–20% reduction in linear

www.landesbioscience.com

rare Diseases

e28341-1

Figure  1. a screen for dominant dNf1 growth defect modifiers. dNf1 mutants are smaller than wild-type flies. to identify modifiers of this phenotype, 486 isogenic deficiencies uncovering ~80% of first and second chromosome genes were crossed into a dNf1 null mutant background, and the length of the resulting pupal cases measured. Confounding factors include that size is a sexually dimorphic phenotype, with males being smaller than females, and that systemic growth is a multifaceted process influenced by environmental factors, such as food availability and temperature. employing strategies to minimize these and other confounding factors, and after eliminating those deficiencies with non-specific effects on growth, candidate dNf1 modifying deficiencies were examined by testing alleles or shrNai lines to identify the responsible modifier genes.

dimensions during all post-embryonic developmental stages.8 Mutants also have a reduced escape response (taking flight upon release), lack a neuropeptideelicited rectifying K+ -current defect at the neuromuscular junction (NMJ), and exhibit circadian arrhythmicity, olfactory associative learning, and memory deficits.8-11 Remarkably, all defects but the circadian arrhythmicity are not particularly sensitive to genetic manipulation of Ras signaling but are suppressed by increasing cAMP/PKA pathway signaling or mimicked by decreasing signaling through the cAMP/ PKA pathway.

While there is little doubt that loss of NF1 affects cAMP/PKA signaling, we and another group have reached contradictory conclusions about the mechanism(s) involved. Yi Zhong and colleagues reported that a C-terminal segment of human neurofibromin (that does not include the RasGAP catalytic domain) is sufficient for NF1/Galpha(S)-dependent neurotransmitter stimulated adenylyl cyclase activation and rescue of the dNf1 growth defect.4 In contrast, we found that expression of a functional dNf1 RasGAP catalytic domain is both necessary and sufficient to restore the cAMP/PKAsensitive growth deficiency. Moreover,

dNf1 expression during the larval growth phase is largely restricted to neurons, and expression of an unrelated Drosophila RasGAP in these cells sufficed to restore normal growth. Finally, although multiple Ras signaling mutants did not dominantly modify dNf1 systemic growth, these mutants also did not reduce the elevated phospho-ERK level in dNf1 larval brain.12 Our conclusion that neuronal Ras/ ERK over-activation is the root cause of the cAMP/PKA-sensitive dNf1 growth defect received further support from subsequent work. Neuronal overexpression of the dAlk receptor tyrosine kinase or of its activating ligand jelly belly (jeb) phenocopied dNf1 growth and learning defects, while genetic or pharmacological attenuation of Jeb/dAlk signaling suppressed both phenotypes. Specifically implicating Ras-stimulated ERK over-activation, this study also found that neuronal expression of a constitutively active ERK mutant phenocopied the dNf1 growth defect.13 To shed further light on dNf1’s role in organismal growth and on the mechanistic links between dNf1 and cAMP/PKA signaling, we recently reported results of an unbiased genetic screen for dominant modifiers of the dNf1 growth defect.14 Our screen analyzed 486 isogenic first and second chromosome deficiencies, each typically uncovering between 1 and 25 genes. The deficiencies, which together uncover close to 80% of first and second chromosome genes, were crossed into the dNf1 null background, and modifying deficiencies were identified by measuring the length of pupal cases (Fig. 1). After eliminating deficiencies that also affect the size of wild-type pupae, responsible modifier genes were identified in crosses with available alleles, or by neuronalor glial-specific RNAi knockdown of candidate genes. Validating the screen, we identified dAlk, its activating ligand jelly belly (jeb), and the dunce (dnc) cAMP phosphodiesterase as dominant suppressors. All three genes had been identified previously as dNf1 phenotypic suppressors.11,13 Earlier work had also established that heat shockinduced expression of a constitutively active murine PKA* catalytic subunit transgene normalized dNf1 size,8 whereas others found reduced brain adenylyl

e28341-2

rare Diseases

Volume 2

cyclase activity upon loss of Drosophila or murine Nf1.15 Thus, we were not surprised to identify the PKA-C1 catalytic subunit as an enhancer, and the PKAR2 regulatory subunit as a yet to be fully confirmed candidate suppressor. Providing mechanistic insights, follow-up experiments indicated that growth regulation by dNf1 and cAMP/PKA likely involves different cells. First, arguing against the idea that PKA suppresses dNf1 defects by attenuating RAS/ERK signaling, we found that widespread or tissue-specific transgenic PKA* expression does not reduce the elevated phospho-ERK level in dNf1 larval brain. Second, whereas only relatively widespread neuronal dNf1 re-expression restored the mutant growth defect,12 in the current study, genetic manipulations that increased cAMP/ PKA signaling in specific parts of the larval ring gland (a neuroendocrine gland analogous to the mammalian pituitary) were sufficient to restore dNf1 growth. By contrast, expressing dNf1 in the ring gland or widespread neuronal expression of a dncRNAi transgene outside of the ring gland had no effect.14 These results argue that dNf1 controls Drosophila growth by non-cell-autonomously affecting cAMP/ PKA signaling in the ring gland (Fig. 2). Whether a similar non-cell-autonomous neuroendocrine mechanism underlies the reduced growth of patients with NF1 or other RASopathies remains to be established. Our screen also identified several dNf1 growth defect suppressors with synaptic functions. Examples include the cAMPcoupled neuronal drosulfakinin receptor CCKLR-17D1, a positive regulator of synaptic growth, and dynamin-associated protein 160 (Dap160), an intersectinrelated scaffold implicated in synaptic vesicle exocytosis and neuroblast proliferation. Because recent work identified a novel dNf1 NMJ overgrowth phenotype,16 we tested whether suppressors identified in our screen also modified the NMJ defect. Suggesting a mechanistic link between both phenotypes, loss-offunction dAlk, Jeb, and CCKLR-D1 alleles reduced the number of NMJ synaptic boutons.14 Studies in C. elegans and Drosophila had previously revealed roles for ALK

Figure 2. Model of dNf1-regulated systemic growth. Neurofibromin functions in neurons of the larval central nervous system to regulate Jeb/dalk-stimulated raS/MeK/erK signaling. In ways that remain poorly understood, excessive neuronal raS/MeK/erK signaling leads to synaptic architecture or neurotransmission defects, which appear causally linked to the reduced growth phenotype. Suggesting a neuroendocrine non-cell-autonomous mechanism, increasing caMp/ pKa signaling in specific segments of the larval brain-associated neuroendocrine ring gland suffices to suppress the dNf1 growth defect. In contrast, only widespread neuronal dNf1 expression restores mutant growth.

orthologs in synapse formation and neuronal differentiation. Thus, work in C. elegans suggested that the F-box protein FSN-1 and the RING finger protein RPM-1 form a ubiquitin ligase complex that controls synapse stability by targeting ALK ortholog T10H9.2/ SCD-2.17 In Drosophila, Jeb and dAlk are both enriched at synapses,18 and function to control neurotransmission strength and synaptic architecture.19 Based on our results, one might speculate that dAlk controls synaptic growth by activating a dNf1-regulated Ras/ERK signal. However, reconciling our data with these other results is less than straightforward, since dNf1 is primarily expressed in neurons and plays its growth-related role in these cells (i.e., presynaptically),14 whereas others concluded that NMJ differentiation involves the activation of postsynaptic (i.e., muscle expressed) dAlk by presynaptically released Jeb.19 Although we can only speculate at this point, one potential explanation is that the growth-related role of Jeb, dAlk, and

dNf1 involves aberrant synaptogenesis between neurons, rather than at the NMJ. It is worth noting in this respect that murine neurofibromin has been implicated in synaptic differentiation,20 and that a recent ultrastructural study found reduced curvature at concave synapses in the hippocampus of Nf1+/− mice.21 Loss of dAlk or Jeb dominantly suppressed dNf1 growth, associative learning, and neuronal ERK overactivation phenotypes, which, together with other results, suggests a role for dAlk as a rate-limiting activator of functionally important dNf1-regulated neuronal RAS/ERK signals.13 The fact that attenuation of dAlk signaling rescues multiple dNf1 defects raises important questions whether NF1-regulated ALK/ RAS/ERK signaling is conserved in man, and whether ALK should be further investigated as a therapeutic target in NF1. Several observations suggest positive answers to both questions. Thus, as we previously found for dAlk and dNf1,13

www.landesbioscience.com

rare Diseases

e28341-3

the expression of Alk and Nf1 in the mouse nervous system overlaps to a large extent.22,23 Supporting an evolutionary conserved functional link between both proteins, mutations that activate ALK or that block the expression of NF1 have both been implicated in neuroblastoma tumorigenesis.24,25 Adding to this indirect evidence, our recent study found that shRNA-mediated suppression of NF1 expression renders human neuroblastoma cells resistant to pharmacological ALK inhibition.14 Specifically, we used two human neuroblastoma lines harboring constitutively active F1174L ALK alleles. Both lines are highly sensitive to pharmacological inhibition of ALK with either NVP-TAE684 or Crizotinib.
References
1. Rauen KA. The RASopathies. Annu Rev Genomics Hum Genet 2013; 14:355-69; PMID:23875798; http://dx.doi.org/10.1146/ annurev-genom-091212-153523 2. Korf BR. Neurofibromatosis. Handb Clin Neurol 2013; 111:333-40; PMID:23622184; http://dx.doi. org/10.1016/B978-0-444-52891-9.00039-7 3. Cichowski K, Jacks T. NF1 tumor suppressor gene function: narrowing the GAP. Cell 2001; 104:593604; PMID:11239415; http://dx.doi.org/10.1016/ S0092-8674(01)00245-8 4. Hannan F, Ho I, Tong JJ, Zhu Y, Nurnberg P, Zhong Y. Effect of neurofibromatosis type I mutations on a novel pathway for adenylyl cyclase activation requiring neurofibromin and Ras. Hum Mol Genet 2006; 15:1087-98; PMID:16513807; http://dx.doi. org/10.1093/hmg/ddl023 5. Ho IS, Hannan F, Guo HF, Hakker I, Zhong Y. Distinct functional domains of neurofibromatosis type 1 regulate immediate versus long-term memory formation. J Neurosci 2007; 27:68527; PMID:17581973; http://dx.doi.org/10.1523/ JNEUROSCI.0933-07.2007 6. Tong JJ, Schriner SE, McCleary D, Day BJ, Wallace DC. Life extension through neurofibromin mitochondrial regulation and antioxidant therapy for neurofibromatosis-1 in Drosophila melanogaster. Nat Genet 2007; 39:476-85; PMID:17369827; http:// dx.doi.org/10.1038/ng2004 7. Easton DF, Ponder MA, Huson SM, Ponder BA. An analysis of variation in expression of neurofibromatosis (NF) type 1 (NF1): evidence for modifying genes. Am J Hum Genet 1993; 53:305-13; PMID:8328449 8. The I, Hannigan GE, Cowley GS, Reginald S, Zhong Y, Gusella JF, Hariharan IK, Bernards A. Rescue of a Drosophila NF1 mutant phenotype by protein kinase A. Science 1997; 276:791-4; PMID:9115203; http:// dx.doi.org/10.1126/science.276.5313.791 9. Guo HF, The I, Hannan F, Bernards A, Zhong Y. Requirement of Drosophila NF1 for activation of adenylyl cyclase by PACAP38-like neuropeptides. Science 1997; 276:795-8; PMID:9115204; http:// dx.doi.org/10.1126/science.276.5313.795 10. Guo HF, Tong J, Hannan F, Luo L, Zhong Y. A neurofibromatosis-1-regulated pathway is required for learning in Drosophila. Nature 2000; 403:895-8; PMID:10706287; http://dx.doi. org/10.1038/35002593

Retroviral shRNA-mediated NF1 knockdown increased the resistance of these cells to both inhibitors, as evidenced by continued growth and sustained MEK/ ERK activation. Further, expression of activated KRAS, BRAF, or MEK transgenes, but not of other Ras effector transgenes, conferred similar resistance to ALK inhibition.14 Two additional observations suggest a potential role for ALK in NF1 tumorigenesis. First, we found that human ALK is expressed in neurofibroma-derived NF1−/− Schwann cells, as well as in cells derived from malignant NF1 tumors. Second, others previously reported that the human ALK ligand midkine is aberrantly expressed in NF1 deficient murine Schwann cells, and
11. Williams JA, Su HS, Bernards A, Field J, Sehgal A. A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK. Science 2001; 293:2251-6; PMID:11567138; http://dx.doi. org/10.1126/science.1063097 12. Walker JA, Tchoudakova AV, McKenney PT, Brill S, Wu D, Cowley GS, Hariharan IK, Bernards A. Reduced growth of Drosophila neurofibromatosis 1 mutants reflects a non-cell-autonomous requirement for GTPase-Activating Protein activity in larval neurons. Genes Dev 2006; 20:331123; PMID:17114577; http://dx.doi.org/10.1101/ gad.1466806 13. Gouzi JY, Moressis A, Walker JA, Apostolopoulou AA, Palmer RH, Bernards A, Skoulakis EM. The receptor tyrosine kinase Alk controls neurofibromin functions in Drosophila growth and learning. PLoS Genet 2011; 7:e1002281; PMID:21949657; http:// dx.doi.org/10.1371/journal.pgen.1002281 14. Walker JA, Gouzi JY, Long JB, Huang S, Maher RC, Xia H, Khalil K, Ray A, Van Vactor D, Bernards R, et al. Genetic and functional studies implicate synaptic overgrowth and ring gland cAMP/PKA signaling defects in the Drosophila melanogaster neurofibromatosis-1 growth deficiency. PLoS Genet 2013; 9:e1003958; PMID:24278035; http://dx.doi. org/10.1371/journal.pgen.1003958 15. Tong J, Hannan F, Zhu Y, Bernards A, Zhong Y. Neurofibromin regulates G protein-stimulated adenylyl cyclase activity. Nat Neurosci 2002; 5:95-6; PMID:11788835; http://dx.doi.org/10.1038/nn792 16. Tsai P-I, Wang M, Kao H-H, Cheng YJ, Walker JA, Chen R-H, Chien C-T. Neurofibromin mediates FAK signaling in confining synapse growth at Drosophila neuromuscular junctions. J Neurosci 2012; 32:1697181; PMID:23175848; http://dx.doi.org/10.1523/ JNEUROSCI.1756-12.2012 17. Liao EH, Hung W, Abrams B, Zhen M. An SCF-like ubiquitin ligase complex that controls presynaptic differentiation. Nature 2004; 430:34550; PMID:15208641; http://dx.doi.org/10.1038/ nature02647 18. Rohrbough J, Broadie K. Anterograde Jelly belly ligand to Alk receptor signaling at developing synapses is regulated by Mind the gap. Development 2010; 137:3523-33; PMID:20876658; http://dx.doi. org/10.1242/dev.047878

acts as a potent mitogen for human NF1 tumor cells.26 Thus, among the various phenotypic suppressors identified in our screen, we feel that ALK should with highest priority be further investigated as a potential therapeutic target in NF1.
Disclosure of Potential Conflict of Interest

No potential conflict of interest was disclosed.
Acknowledgments

We would like to thank our co-authors. Work in our laboratory was supported by grants from the NIH, the Department of Defense Neurofibromatosis Research Program, and the Children’s Tumor Foundation.
19. Rohrbough J, Kent KS, Broadie K, Weiss JB. Jelly Belly trans-synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture. Dev Neurobiol 2013; 73:189208; PMID:22949158; http://dx.doi.org/10.1002/ dneu.22056 20. Hsueh YP. Neurofibromin signaling and synapses. J Biomed Sci 2007; 14:461-6; PMID:17370141; http://dx.doi.org/10.1007/s11373-007-9158-2 21. Armstrong BC, Le Boutillier JC, Petit TL. Ultrastructural synaptic changes associated with neurofibromatosis type 1: a quantitative analysis of hippocampal region CA1 in a Nf1(+/-) mouse model. Synapse 2012; 66:246-55; PMID:22121000; http:// dx.doi.org/10.1002/syn.21507 22. Daston MM, Ratner N. Neurofibromin, a predominantly neuronal GTPase activating protein in the adult, is ubiquitously expressed during development. Dev Dyn 1992; 195:21626; PMID:1301085; http://dx.doi.org/10.1002/ aja.1001950307 23. Iwahara T, Fujimoto J, Wen D, Cupples R, Bucay N, Arakawa T, Mori S, Ratzkin B, Yamamoto T. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 1997; 14:439-49; PMID:9053841; http://dx.doi.org/10.1038/sj.onc.1200849 24. Hölzel M, Huang S, Koster J, Ora I, Lakeman A, Caron H, Nijkamp W, Xie J, Callens T, Asgharzadeh S, et al. NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome. Cell 2010; 142:218-29; PMID:20655465; http://dx.doi.org/10.1016/j.cell.2010.06.004 25. Mano H. ALKoma: a cancer subtype with a shared target. Cancer Discov 2012; 2:495-502; PMID:22614325; http://dx.doi.org/10.1158/21598290.CD-12-0009 26. Mashour GA, Ratner N, Khan GA, Wang HL, Martuza RL, Kurtz A. The angiogenic factor midkine is aberrantly expressed in NF1-deficient Schwann cells and is a mitogen for neurofibroma-derived cells. Oncogene 2001; 20:97-105; PMID:11244508; http://dx.doi.org/10.1038/sj.onc.1204026

e28341-4

rare Diseases

Volume 2