Genetic and Functional Studies Implicate Synaptic Overgrowth and Ring Gland cAMP/PKA Signaling Defects in the Drosophila melanogaster Neurofibromatosis-1 Growth Deficiency

Abstract

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 melanogaster 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 we screened 486 1st and 2nd chromosome deficiencies that uncover >80% of annotated genes 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, we show 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 our previous contention that ALK might be a therapeutic target in NF1, we report 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.

Physical interaction between neurofibromin and serotonin 5-HT 6 receptor promotes receptor constitutive activity

“…Whether the neurofibromin PH domain associated with 5-HT 6 receptor favors recruitment of Gs protein remains to be explored. A recent study has shown that the regulation by neurofibromin of cAMP signaling requires Ras activation and operates through the activation of atypical PKC zeta (28), whereas other studies performed in Drosophila suggest that neurofibromin controls cAMP production in an Ras-independent manner (19,29). Our results showing that expression of PH domain alone is sufficient to rescue normal 5-HT 6 receptor constitutive activity in neurofibromin-deficient cells are consistent with an Ras-independent mechanism.…”

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

“…Su(H)‐LacZ [ Su(H) GBE ‐LacZ , (Furriols & Bray, 2001)], esg‐Gal4 NP7397 , UAS‐GFP , tub‐Gal80 TS chromosome (gift from J. de Navascués), Ret‐Gal4 (Walker et al , 2013), wg KO ‐Gal4 (Alexandre et al , 2014; gift from Luis‐Alberto Baena‐Lopez).…”

ADrosophilascreen identifies neurofibromatosis-1 genetic modifiers involved in systemic and synaptic growth

“…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 dnc RNAi 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.…”

“…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.…”

“…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.…”