MAPK3 at the Autism-Linked Human 16p11.2 Locus Influences Precise Synaptic Target Selection at Drosophila Larval Neuromuscular Junctions

  • Park, Sang Mee (Department of Oral Pathology and BK21Plus Project, School of Dentistry, Pusan National University) ;
  • Park, Hae Ryoun (Department of Oral Pathology and BK21Plus Project, School of Dentistry, Pusan National University) ;
  • Lee, Ji Hye (Department of Oral Pathology and BK21Plus Project, School of Dentistry, Pusan National University)
  • Received : 2016.12.13
  • Accepted : 2017.01.23
  • Published : 2017.02.28


Proper synaptic function in neural circuits requires precise pairings between correct pre- and post-synaptic partners. Errors in this process may underlie development of neuropsychiatric disorders, such as autism spectrum disorder (ASD). Development of ASD can be influenced by genetic factors, including copy number variations (CNVs). In this study, we focused on a CNV occurring at the 16p11.2 locus in the human genome and investigated potential defects in synaptic connectivity caused by reduced activities of genes located in this region at Drosophila larval neuromuscular junctions, a well-established model synapse with stereotypic synaptic structures. A mutation of rolled, a Drosophila homolog of human mitogen-activated protein kinase 3 (MAPK3) at the 16p11.2 locus, caused ectopic innervation of axonal branches and their abnormal defasciculation. The specificity of these phenotypes was confirmed by expression of wild-type rolled in the mutant background. Albeit to a lesser extent, we also observed ectopic innervation patterns in mutants defective in Cdk2, Gq, and Gp93, all of which were expected to interact with Rolled MAPK3. A further genetic analysis in double heterozygous combinations revealed a synergistic interaction between rolled and Gp93. In addition, results from RT-qPCR analyses indicated consistently reduced rolled mRNA levels in Cdk2, Gq, and Gp93 mutants. Taken together, these data suggest a central role of MAPK3 in regulating the precise targeting of presynaptic axons to proper postsynaptic targets, a critical step that may be altered significantly in ASD.


16p11.2;autism;copy number variations;Drosophila;MAPK3


Supported by : Pusan National University


  1. Atwood, H., Govind, C., and Wu, C.F. (1993). Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae. J. Neurobiol. 24, 1008-1024.
  2. Banerjee, S., Venkatesan, A., and Bhat, M.A. (2016). Neurexin, Neuroligin and Wishful Thinking coordinate synaptic cytoarchite- cture and growth at neuromuscular junctions. Mol. Cell Neurosci. 78, 9-24.
  3. Bauman, M.L., and Kemper, T.L. (2005). Neuroanatomic observations of the brain in autism: a review and future directions. Int. J. Dev. Neurosci. 23, 183-187.
  4. Beck, E.S., Gasque, G., Imlach, W.L., Jiao, W., Choi, B.J., Wu, P.-S., Kraushar, M.L., and McCabe, B.D. (2012). Regulation of Fasciclin II and synaptic terminal development by the splicing factor beag. J. Neurosci. 32, 7058-7073.
  5. Betancur, C., Sakurai, T., and Buxbaum, J.D. (2009). The emerging role of synaptic cell-adhesion pathways in the pathogenesis of autism spectrum disorders. Trends Neurosci. 32, 402-412.
  6. Biggs, W.H., and Zipursky, S.L. (1992). Primary structure, expression, and signal-dependent tyrosine phosphorylation of a Drosophila homolog of extracellular signal-regulated kinase. Proc. Natl. Acad. Sci. USA 89, 6295-6299.
  7. Blaker-Lee, A., Gupta, S., McCammon, J.M., De Rienzo, G., and Sive, H. (2012). Zebrafish homologs of genes within 16p11.2, a genomic region associated with brain disorders, are active during brain development, and include two deletion dosage sensor genes. Dis. Model. Mech. 5, 834-851.
  8. Blanchard, D.A., Mouhamad, S., Auffredou, M.-T., Pesty, A., Bertoglio, J., Leca, G., and Vazquez, A. (2000). Cdk2 associates with MAP kinase in vivo and its nuclear translocation is dependent on MAP kinase activation in IL-2-dependent Kit 225 T lymphocytes. Oncogene 19, 4184-4189.
  9. Boulton, T.G., and Cobb, M.H. (1991). Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. Cell Regul. 2, 357-371.
  10. Boulton, T.G., Nye, S.H., Robbins, D.J., Ip, N.Y., Radziejewska, E., Morgenbesser, S.D., DePinho, R.A., Panayotatos, N., Cobb, M.H., and Yancopoulos, G.D. (1991). ERKs: a family of proteinserine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65, 663-675.
  11. Bourgeron, T. (2015). From the genetic architecture to synaptic plasticity in autism spectrum disorder. Nat Rev Neurosci 16, 551-563.
  12. Brand, A.H., and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
  13. Chen, J., Yu, S., Fu, Y., and Li, X. (2014). Synaptic proteins and receptors defects in autism spectrum disorders. Front. Cell. Neurosci. 8, 276.
  14. Christensen, R., Shao, Z., and Colon-Ramos, D.A. (2013). The cell biology of synaptic specificity during development. Curr. Opin. Neurobiol. 23, 1018-1026.
  15. Abrahams, B.S., and Geschwind, D.H. (2008). Advances in autism genetics: on the threshold of a new neurobiology. Nat. Rev. Genet. 9, 341-355.
  16. Amaral, D.G., Schumann, C.M., and Nordahl, C.W. (2008). Neuroanatomy of autism. Trends Neurosci. 31, 137-145.
  17. Ansa-Addo, E.A., Thaxton, J., Hong, F., Wu, B.X., Zhang, Y., Fugle, C.W., Metelli, A., Riesenberg, B., Williams, K., Gewirth, D.T., et al. (2016). Clients and oncogenic roles of molecular chaperone gp96/grp94. Curr. Top Med. Chem. 16, 2765-2778.
  18. Arnold, S.E. (1999). Neurodevelopmental abnormalities in schizophrenia: insights from neuropathology. Dev. Psychopathol. 11, 439-456.
  19. Courchesne, E., Mouton, P.R., Calhoun, M.E., Semendeferi, K., Ahrens-Barbeau, C., Hallet, M.J., Barnes, C.C., and Pierce, K. (2011). Neuron number and size in prefrontal cortex of children with autism. JAMA 306, 2001-2010.
  20. Ecker, C., Suckling, J., Deoni, S.C., Lombardo, M.V., Bullmore, E.T., Baron-Cohen, S., Catani, M., Jezzard, P., Barnes, A., Bailey, A.J., et al. (2012). Brain anatomy and its relationship to behavior in adults with autism spectrum disorder: a multicenter magnetic resonance imaging study. Arch. Gen. Psychiatry 69, 195-209.
  21. Friedman, A.A., Tucker, G., Singh, R., Yan, D., Vinayagam, A., Hu, Y., Binari, R., Hong, P., Sun, X., and Porto, M. (2011). Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling. Sci. Signal. 4, rs10.
  22. Golzio, C., Willer, J., Talkowski, M.E., Oh, E.C., Taniguchi, Y., Jacquemont, S., Reymond, A., Sun, M., Sawa, A., and Gusella, J.F. (2012). KCTD13 is a major driver of mirrored neuroanatomical phenotypes of the 16p11. 2 copy number variant. Nature 485, 363-367.
  23. Gorczyca, M., Augart, C., and Budnik, V. (1993). Insulin-like receptor and insulin-like peptide are localized at neuromuscular junctions in Drosophila. J. Neurosci. 13, 3692-3704.
  24. Gramates, L.S., and Budnik, V. (1999). Assembly and maturation of the Drosophila larval neuromuscular junction. Int. Rev. Neurobiol. 43, 93-117.
  25. Gregorio, S.P., Sallet, P.C., Do, K.A., Lin, E., Gattaz, W.F., and Dias-Neto, E. (2009). Polymorphisms in genes involved in neurodevelopment may be associated with altered brain morphology in schizophrenia: preliminary evidence. Psychiatry Res 165, 1-9.
  26. Henry, S., Pfenninger, K.H., Mott, J.L., and Granholm, A.-C. (1999). Anatomical distribution of glycoprotein 93 (gp93) on nerve fibers during rat brain development. Cell Tissue Res. 297, 67-79.
  27. Hernandez, R., Garcia, F., Encio, I., and De Miguel, C. (2004). Promoter analysis of the human p44 mitogen-activated protein kinase gene (MAPK3): transcriptional repression under nonproliferating conditions. Genomics 84, 222-226.
  28. Hoang, B., and Chiba, A. (2001). Single-cell analysis of Drosophila larval neuromuscular synapses. Dev. Biol. 229, 55-70.
  29. Horev, G., Ellegood, J., Lerch, J.P., Son, Y.-E.E., Muthuswamy, L., Vogel, H., Krieger, A.M., Buja, A., Henkelman, R.M., and Wigler, M. (2011). Dosage-dependent phenotypes in models of 16p11. 2 lesions found in autism. Proc. Natl. Acad. Sci. USA 108, 17076-17081.
  30. Jacobson, J.D., Ellerbeck, K.A., Kelly, K.A., Fleming, K.K., Jamison, T.R., Coffey, C.W., Smith, C.M., Reese, R.M., and Sands, S.A. (2014). Evidence for alterations in stimulatory G proteins and oxytocin levels in children with autism. Psychoneuroendocrinology 40, 159-169.
  31. Johansen, J., Halpern, M.E., Johansen, K.M., and Keshishian, H. (1989). Stereotypic morphology of glutamatergic synapses on identified muscle cells of Drosophila larvae. J. Neurosci. 9, 710-725.
  32. John, J.P., Thirunavukkarasu, P., Halahalli, H.N., Purushottam, M., and Jain, S. (2015). A systematic review of the effect of genes mediating neurodevelopment and neurotransmission on brain morphology: Focus on schizophrenia. Neurol. Psychiatry Brain Res. 21, 1-26.
  33. Kamiya, A., Kubo, K., Tomoda, T., Takaki, M., Youn, R., Ozeki, Y., Sawamura, N., Park, U., Kudo, C., Okawa, M., et al. (2005). A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development. Nat. Cell Biol. 7, 1167-1178.
  34. Koh, Y.-H., Gorczyca, M., and Budnik, V. (2002). The Ras1-Mitogen-Activated Protein Kinase Signal Transduction Pathway Regulates Synaptic Plasticity through Fasciclin II-Mediated Cell Adhesion. J. Neurosci. 22, 2496-2504.
  35. Kolomeets, N.S., Orlovskaya, D.D., Rachmanova, V.I., and Uranova, N.A. (2005). Ultrastructural alterations in hippocampal mossy fiber synapses in schizophrenia: a postmortem morphometric study. Synapse 57, 47-55.
  36. Kumar, R.A., KaraMohamed, S., Sudi, J., Conrad, D.F., Brune, C., Badner, J.A., Gilliam, T.C., Nowak, N.J., Cook, E.H., and Dobyns, W.B. (2008). Recurrent 16p11. 2 microdeletions in autism. Hum. Mol. Genet. 17, 628-638.
  37. Law, A.J., Weickert, C.S., Hyde, T.M., Kleinman, J.E., and Harrison, P.J. (2014). Reduced spinophilin but not microtubule-associated protein 2 expression in the hippocampal formation in schizophrenia and mood disorders: molecular evidence for a pathology of dendritic spines. Am. J. Psychiatry 161, 1848-1855.
  38. Lee, J., and Wu, C.F. (2010). Orchestration of stepwise synaptic growth by $K^+$ and $Ca^{2+}$ channels in Drosophila. J. Neurosci. 30, 15821-15833.
  39. Levitt, P., Ebert, P., Mirnics, K., Nimgaonkar, V.L., and Lewis, D.A. (2006). Making the case for a candidate vulnerability gene in schizophrenia: Convergent evidence for regulator of G-protein signaling 4 (RGS4). Biol. Psychiatry 60, 534-537.
  40. Li, H., Quiroga, S., and Pfenninger, K.H. (1992). Variable membrane glycoproteins in different growth cone populations. J. Neurosci. 12, 2393-2402.
  41. Lin, D.M., Fetter, R.D., Kopczynski, C., Grenningloh, G., and Goodman, C.S. (1994). Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation. Neuron 13, 1055-1069.
  42. Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2- ${\Delta}{\Delta}CT$ method. Methods 25, 402-408.
  43. Marshall, C.R., Noor, A., Vincent, J.B., Lionel, A.C., Feuk, L., Skaug, J., Shago, M., Moessner, R., Pinto, D., and Ren, Y. (2008). Structural variation of chromosomes in autism spectrum disorder. Am. J. Hum. Genet. 82, 477-488.
  44. Menon, K.P., Carrillo, R.A., and Zinn, K. (2013). Development and plasticity of the Drosophila larval neuromuscular junction. Wiley Interdiscip Rev. Dev. Biol. 2, 647-670.
  45. Miyazaki, T., Hashimoto, K., Uda, A., Sakagami, H., Nakamura, Y., Saito, S.-y., Nishi, M., Kume, H., Tohgo, A., and Kaneko, I. (2006). Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family. FEBS Lett. 580, 4057-4064.
  46. Park, S.M., Littleton, J.T., Park, H.R., and Lee, J.H. (2016). Drosophila Homolog of Human KIF22 at the Autism-Linked 16p11.2 Loci influences synaptic connectivity at larval neuromuscular junctions. Exp. Neurobiol. 25, 33-39.
  47. Portmann, T., Yang, M., Mao, R., Panagiotakos, G., Ellegood, J., Dolen, G., Bader, P.L., Grueter, Brad A., Goold, C., Fisher, E., et al. (2014). Behavioral abnormalities and circuit defects in the basal ganglia of a mouse model of 16p11.2 deletion syndrome. Cell Rep. 7, 1077-1092.
  48. Pucilowska, J., Vithayathil, J., Tavares, E.J., Kelly, C., Karlo, J.C., and Landreth, G.E. (2015). The 16p11. 2 deletion mouse model of autism exhibits altered cortical progenitor proliferation and brain cytoarchitecture linked to the ERK MAPK pathway. J. Neurosci. 35, 3190-3200.
  49. Redies, C., Hertel, N., and Hubner, C.A. (2012). Cadherins and neuropsychiatric disorders. Brain Res. 1470, 130-144.
  50. Reiss, A.L., Feinstein, C., and Rosenbaum, K.N. (1986). Autism and genetic disorders. Schizophrenia Bull. 12, 724.
  51. Sanchez-Fernandez, G., Cabezudo, S., Garcia-Hoz, C., Beninca, C., Aragay, A.M., Mayor, F., Jr., and Ribas, C. (2014). Galphaq signalling: the new and the old. Cell Signal. 26, 833-848.
  52. Schuster, C.M., Davis, G.W., Fetter, R.D., and Goodman, C.S. (1996a). Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth. Neuron 17, 641-654.
  53. Schuster, C.M., Davis, G.W., Fetter, R.D., and Goodman, C.S. (1996b). Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity. Neuron 17, 655-667.
  54. Sebat, J., Lakshmi, B., Malhotra, D., Troge, J., Lese-Martin, C., Walsh, T., Yamrom, B., Yoon, S., Krasnitz, A., and Kendall, J. (2007). Strong association of de novo copy number mutations with autism. Science 316, 445-449.
  55. Steen, R.G., Mull, C., McClure, R., Hamer, R.M., and Lieberman, J.A. (2006). Brain volume in first-episode schizophrenia: systematic review and meta-analysis of magnetic resonance imaging studies. Br. J. Psychiatry 188, 510-518.
  56. Stockmeier, C.A., Mahajan, G.J., Konick, L.C., Overholser, J.C., Jurjus, G.J., Meltzer, H.Y., Uylings, H.B.M., Friedman, L., and Rajkowska, G. (2004). Cellular changes in the postmortem hippocampus in major depression. Biol. Psychiatry 56, 640-650.
  57. Sweatt, J.D. (2001). The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory. J. Neurochem. 76, 1-10.
  58. Tessier-Lavigne, M., and Goodman, C.S. (1996). The molecular biology of axon guidance. Science 274, 1123-1133.
  59. Tsai, L.H., Lees, E., Faha, B., Harlow, E., and Riabowol, K. (1993). The cdk2 kinase is required for the G1-to-S transition in mammalian cells. Oncogene 8, 1593-1602.
  60. Vithayathil, J., Pucilowska, J., Goodnough, L.H., Atit, R.P., and Landreth, G.E. (2015). Dentate gyrus development requires ERK activity to maintain progenitor population and MAPK pathway feedback regulation. J. Neurosci. 35, 6836-6848.
  61. Wang, B., Gao, Y., Xiao, Z., Chen, B., Han, J., Zhang, J., Wang, X., and Dai, J. (2009). Erk1/2 promotes proliferation and inhibits neuronal differentiation of neural stem cells. Neurosci. Lett. 461, 252-257.
  62. Weiss, L.A., Shen, Y., Korn, J.M., Arking, D.E., Miller, D.T., Fossdal, R., Saemundsen, E., Stefansson, H., Ferreira, M.A., and Green, T. (2008). Association between microdeletion and microduplication at 16p11. 2 and autism. N Engl. J. Med. 358, 667-675.
  63. Yoshida, T., McCarley, R.W., Nakamura, M., Lee, K., Koo, M.-S., Bouix, S., Salisbury, D.F., Morra, L., Shenton, M.E., and Niznikiewicz, M.A. (2009). A prospective longitudinal volumetric MRI study of superior temporal gyrus gray matter and amygdala-hippocampal complex in chronic schizophrenia. Schizophrenia Res. 113, 84-94.
  64. Zoghbi, H.Y., and Bear, M.F. (2012). Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities. Cold Spring Harb Perspect Biol. 4.