Role of MAGUK complexes in synaptic function

Synaptic transmission relies on the proper spatial organization of protein complexes and for this,  among the multiple proteins that compose the synapse the group of scaffold proteins play a fundamental role in the organization of membrane subdomains. Scaffold proteins are molecules with multiple protein-protein interaction domains that allow the formation of protein complexes in cell domains. The Membrane-Associated Guanylate Kinase (MAGUK) family of scaffolding proteins and in particular the Synapse Associated Proteins (SAP) subfamily, is extensively expressed in the brain and links synaptic proteins to signal transduction cascades, the cytoskeleton and the endocytic machinery

In vertebrates, SAPs PSD95/SAP90, SAP102/NEDLG, SAP97/hDLG, and PSD93/Chapsyn-110 are thought to play key roles in synapse assembly and plasticity in vivo evidence for these roles has been scarce, likely due to functional redundancy. Drosophila contains a single SAP gene, dlg, with functions in epithelial development and in the development and function of the neural tissue and particularly the synapses including the larval neuromuscular junction (NMJ). In dlg mutants, all gene products are severely decreased, which leads to early pupal lethality, and thus precludes their analysis beyond this stage.

We have characterized a variety of products of the dlg locus, which are expressed in epithelial, neuronal and muscle tissue. Mutants that have a decreased expression of all splice variants are lethal at early larvae stage impairing the study of the role of this gene in brain processes posterior to the embryonic or early larvae stage. Through the movilization of genetic transposable elements we have obtained specific null mutants for the main splice products of the dlg gene. Importantly the null mutants for the neuronal isoform are viable, allowing for the first time to study the role of dlg in synaptic function in complex processes such as memory, learning and behavior.

Figure: Generation of isoform-specific dlg mutants. (A) Map of the dlg genomic region indicating the position of the P-element insertions used to generate the mutants (triangles at top) and the regions deleted in dlgS97138, dlgS975, and dlgA40.2 (see text for details), exons are numbered as in Mendoza et al 2003, 1 to 24 are translated exons, A and B are untranslated exons. (B) Exonic composition (as above) and protein domains of DLGS97 and DLGA variants. The regions recognized by DLGS97N and DLGPDZ antibodies are underlined (note that the DLGPDZ antibody recognizes both variants). (C) Whole-embryo DLGS97N immunocytochemistry in wild type and dlgS97 mutants, showing the lack of signal in the mutants. (D) Embryonic ventral nerve cords double-stained with DLGS97N (green) and DLGPDZ (red) antibodies, showing that DLGPDZ immunoreactivity in dlgS97 mutants is not drastically reduced. (E) Western blot of body wall muscle protein extracts in wild type, dlgA and dlgS97 mutants, sequentially probed with anti-DLGPDZ, anti-DLGS97N, and anti-Tubulin. Molecular weights are shown to the left in kDa. Calibration bar in C is 20 µm.

1. Role of the dlg gene in the function of glutamatergic synapses

Larval neuromuscular junction

The larval neuromuscular junction is a glutamatergic synapse that shares several characteristics with mammalian central nervous system synapses. This characteristic plus the accesibility and the genetics tools available makes the NMJ an attractive model for neurobiology.

We study the localization and abundance of synaptic proteins in different genetic backgrounds. In addition by the use of electrophysiological techniques such as voltage clamp and current clamp we are studying the synaptic transmission defects in the synaptic mutants. We are correlating the physiology with the morphological defects of the synapses observed by confocal and electronic microscopy.

2. Behavioral studies in synaptic protein mutants

The fruit fly Drosophila melanogaster is a complex organism that posses innate and adaptive behaviors that can be studied from a genetic approach. We have found that complex behaviors such as courtship, phototactism and circadian rhythm are altered in dlg mutants. Simple behaviors are not different in the mutants at room temperature but they show dramatic alterations at temperatures that impose an additional stress to the synapse. We are carrying out studies on simple behaviors such as locomotion and olfaction but we are also in the process of implementing the paradigms to tests learning and memory in order to test the role of dlg and other synaptic proteins of the complex in these processes. We will correlate these studies with the synaptic defects that we are characterizing in the NMJ.

Figure 3: Behavioral analysis and ERG recordings of dlgS97 mutants. (A) Climbing and benzaldehyde odor avoidance assay, showing no significant differences between mutants and controls. Bars ± SEM represent the average of 9 to 21 (climbing score) and 11 to 19 (odor detection) independent experiments. (B) Phototaxis assay showing the percentage of flies remaining in the dark. Left: comparison between controls, dlgS97 mutants, and inaD mutants showing that dlgS97 mutants have phototaxis defects that are not statistically different from inaD mutants and different from controls. Each point corresponds to the average of three independent experiments ± SEM. Right: the mutant defects in phototaxis are completely rescued by neuronal expression of DLGS97, but only partially rescued by the expression of DLGA. (C) ERGs from wild-type, dlgS975, and dlgS97138 flies. The stimulus was a 1-second pulse of white light (400-700 nm), log I/I 0 = -4 for the first light stimulus and log I/I 0 = -2 for the second light stimulus; I 0 = 6600 lux, at the time indicated beneath the recordings. Note the lack of differences between wild type and mutants. (D) Courtship index of individual control and dlgS975 males showing that courtship behavior is defective in the mutant, and that this phenotype can be rescued by neuronal DLGS97 but not by DLGA expression. Horizontal lines represent the median of each group. Levels of significance were determined by a 2-tail Student t-test in A and B and by a Wilcoxon-Mann-Whitney test in D (* P<0.05 and ** P<0.01).

3. Identification of the regulatory genomic regions of the dlg gene