Learning Something ORIGINal at the Drosophila Neuromuscular Junction

Genetic screens seek to identify molecules based on phenotype, without regard to the expectations of the experimenter. A classic example is the screen by Nusslein-Volhard and Weischaus 1980xNusslein-Volhard, C. and Weischaus, E. Nature. 1980; 287: 795–801Crossref | PubMed | Scopus (2062)See all ReferencesNusslein-Volhard and Weischaus 1980 that identified most of the genes required for embryonic pattern formation. Such an approach is being applied to a question of special interest to neurobiologists—what are the molecules and mechanisms required for learning and memory? Drosophila can be taught to associate an odor with a footshock, and single gene mutants have been identified that are defective in either learning or remembering this task. Most such mutants show defects in two areas, brain development and synaptic plasticity (Dubnau and Tully 1998xDubnau, J. and Tully, T. Annu. Rev. Neurosci. 1998; 21: 407–444Crossref | PubMed | Scopus (265)See all ReferencesDubnau and Tully 1998). Previously identified plasticity mutants conform to expectations, encoding proteins involved in signal transduction. Now, however, genetics presents us with a surprise. The gene for the learning mutant latheo (lat) encodes a component of the origin replication complex (ORC), which is important for DNA replication, but the protein is also present at the synapse and regulates synaptic function and plasticity.latheo (Greek for “to cause a person not to know”) was identified in a genetic screen for Drosophila mutants with defects in associative learning and memory (Boynton and Tully 1992xBoynton, S. and Tully, T. Genetics. 1992; 131: 655–672PubMedSee all ReferencesBoynton and Tully 1992). In this issue of Neuron, a collaboration between the Tully and Dutta groups presents the cloning of lat and the characterization of its biochemical function (Pinto et al. 1999xPinto, S., Quintana, D.G., Smith, P., Mihalek, R.M., Hou, Z.-H., Boynton, S., Jones, C.J., Hendricks, M., Velinzon, K., Wohlschlegel, J.A. et al. Neuron. 1999; 23: 45–54Abstract | Full Text | Full Text PDF | PubMed | Scopus (91)See all ReferencesPinto et al. 1999). Tully and colleagues identify the transcript disrupted in lat and find that it encodes a novel gene homologous to a human expressed sequence tag (EST). In independent experiments, Dutta and colleagues have isolated new constituents of the human ORC, a group of proteins essential for DNA replication (Rowles and Blow 1997xRowles, A. and Blow, J.J. Curr. Opin. Genet. Dev. 1997; 7: 152–157Crossref | PubMed | Scopus (61)See all ReferencesRowles and Blow 1997). Pinto, Quintana, and colleagues 1999xPinto, S., Quintana, D.G., Smith, P., Mihalek, R.M., Hou, Z.-H., Boynton, S., Jones, C.J., Hendricks, M., Velinzon, K., Wohlschlegel, J.A. et al. Neuron. 1999; 23: 45–54Abstract | Full Text | Full Text PDF | PubMed | Scopus (91)See all ReferencesPinto, Quintana, and colleagues 1999 find that an ORC protein is encoded by the human lat homolog, and show that human and Drosophila latheo (LAT) proteins biochemically associate with the ORC and are homologous to the yeast protein ORC3.Why was a DNA replication protein identified in a screen for learning and memory mutants? A weak allele of lat shows the learning and memory defect, while stronger alleles are pupal lethal. Pinto et al. 1999xPinto, S., Quintana, D.G., Smith, P., Mihalek, R.M., Hou, Z.-H., Boynton, S., Jones, C.J., Hendricks, M., Velinzon, K., Wohlschlegel, J.A. et al. Neuron. 1999; 23: 45–54Abstract | Full Text | Full Text PDF | PubMed | Scopus (91)See all ReferencesPinto et al. 1999 demonstrate that null mutations of lat have severe defects in cell proliferation; the mutants lack imaginal discs, proliferating cell clusters that form much of the adult body. In addition, neuroblast proliferation is disrupted, so these mutants have small brains. The mushroom bodies, structures crucial for olfactory learning (Heisenberg et al. 1985xHeisenberg, M., Borst, A., Wagner, S., and Byers, D. J. Neurogenet. 1985; 2: 1–30Crossref | PubMed | Scopus (2)See all ReferencesHeisenberg et al. 1985), are reduced in both weak and strong lat mutants, providing a satisfying explanation for lat’s learning and memory defect.This, however, is not the entire story. Many Drosophila learning and memory mutants have morphological and functional synaptic defects that can be assayed at the neuromuscular junction (NMJ) (Dubnau and Tully 1998xDubnau, J. and Tully, T. Annu. Rev. Neurosci. 1998; 21: 407–444Crossref | PubMed | Scopus (265)See all ReferencesDubnau and Tully 1998). In this issue of Neuron, work from Broadie and colleagues (Rohrbough et al. 1999xRohrbough, J., Pinto, S., Mihalek, R.M., Tully, T., and Broadie, K. Neuron. 1999; 23: 55–70Abstract | Full Text | Full Text PDF | PubMed | Scopus (69)See all ReferencesRohrbough et al. 1999) analyzes the synaptic phenotype of lat mutants. The most unexpected result is that the LAT protein is localized to synaptic boutons of the fly NMJ. While the staining intensity varies at different synaptic sites, the protein appears to be present at all neuromuscular synapses. Double staining with a variety of synaptic markers suggests that the protein is present in the presynaptic terminal. Having demonstrated that LAT is present at the NMJ, the authors characterize synaptic structure and function in lat mutants. They find only subtle alterations of synaptic morphology but a profound change in the function of the synapse.latheo mutants show a large increase in the amplitude of evoked synaptic events, with no change in the postsynaptic responsiveness to transmitter. Hence, in the mutant many more vesicles are released when the nerve is stimulated. In addition to this alteration of basal synaptic transmission, lat has defects in synaptic plasticity. The Drosophila NMJ exhibits many forms of activity-dependent synaptic plasticity including paired-pulse facilitation, augmentation, and posttetanic potentiation (Zhong and Wu 1991xZhong, Y. and Wu, C.F. Science. 1991; 251: 198–201Crossref | PubMedSee all ReferencesZhong and Wu 1991). All of these plasticity mechanisms are disrupted in lat. These defects are not merely a result of the increase in basal transmission in the mutant. When basal transmission is decreased in the mutant to wild-type levels (by decreasing the extracellular calcium concentration), there is still an impairment in all forms of activity-dependent plasticity.The involvement of a DNA replication protein in functional synaptic plasticity is, to put it mildly, unexpected, and leaves us with three interpretations of the synaptic phenotype of latheo mutants. First, the synaptic defects may be downstream of LAT’s role in the nucleus, and the presence of LAT at the synapse may be a fluke. Second, the ORC has two functions, one in the nucleus and one at the synapse. Third, LAT has two functions, one as a part of the ORC and a second at the synapse. Fortunately, the reagents exist to test these three models. LAT biochemically interacts with another subunit of the Drosophila ORC, DmORC2. Mutants for DmORC2 show an identical proliferation phenotype to lat, with late larval lethality, missing imaginal discs, and defective cell proliferation (3xGatti, M. and Baker, B.S. Genes Dev. 1989; 3: 438–453Crossref | PubMedSee all References, 6xLandis, G., Kelley, R., Spradling, A.C., and Tower, J. Proc. Natl. Acad. Sci. USA. 1997; 94: 3888–3892Crossref | PubMed | Scopus (101)See all References). In addition, antibodies have been generated to DmORC2 (Gossen et al. 1995xGossen, M., Pak, D.T.S., Hansen, S.K., Acharya, J.K., and Botchan, M.R. Science. 1995; 270: 1674–1677Crossref | PubMedSee all ReferencesGossen et al. 1995). Future experiments can address whether DmORC2 mutants have a synaptic phenotype and if the protein is synaptically localized. If DmORC2 mutants do show the latheo synaptic phenotype but DmORC2 is not present at the synapse, this would suggest that the synaptic defects are downstream of the ORC’s role in replication. If DmORC2 is at the NMJ, this would lead to the exciting hypothesis that the ORC has a second function at the synapse. Finally, if DmORC2 mutants have no synaptic phenotype, this would argue that LAT has a second function at the synapse. Regardless of the final explanation for the unexpected results with LAT, genetics has served its purpose in calling attention to a molecule that is not a usual suspect at the synapse.How, in the end, should we think about the learning phenotype of latheo mutants? Is it a result of defective mushroom body development, impaired synaptic plasticity, or both? A satisfying answer must await the identification of mutants that separate the roles of lat at the chromosome and the synapse, or the creation of conditional mutants that rescue only the developmental defects. In the meantime, these two papers in Neuron may inspire neurobiologists to head to the library for a refresher course on DNA replication.