Glycogen synthase kinase 3, circadian rhythms, and bipolar disorder: a molecular link in the therapeutic action of lithium

BACKGROUND: Bipolar disorder (BPD) is a widespread condition characterized by recurring states of mania and depression. Lithium, a direct inhibitor of glycogen synthase kinase 3 (GSK3) activity, and a mainstay in BPD therapeutics, has been proposed to target GSK3 as a mechanism of mood stabilization. In addition to mood imbalances, patients with BPD often suffer from circadian disturbances. GSK3, an essential kinase with widespread roles in development, cell survival, and metabolism has been demonstrated to be an essential component of the Drosophila circadian clock. We sought to investigate the role of GSK3 in the mammalian clock mechanism, as a possible mediator of lithium's therapeutic effects. METHODS: GSK3 activity was decreased in mouse embryonic fibroblasts (MEFs) genetically and pharmacologically, and changes in the cyclical expression of core clock genes – mPer2 in particular – were examined. RESULTS: We demonstrate that genetic depletion of GSK3 in synchronized oscillating MEFs results in a significant delay in the periodicity of the endogenous clock mechanism, particularly in the cycling period of mPer2. Furthermore, we demonstrate that pharmacological inhibition of GSK3 activity by kenpaullone, a known antagonist of GSK3 activity, as well as by lithium, a direct inhibitor of GSK3 and the most common treatment for BPD, induces a phase delay in mPer2 transcription that resembles the effect observed with GSK3 knockdown. CONCLUSION: These results confirm GSK3 as a plausible target of lithium action in BPD therapeutics, and suggest the circadian clock mechanism as a significant modulator of lithium's clinical benefits

Spatio-temporal regulation and activity of segmentation genes in Drosophila melanogaster

The segmentation genes of Drosophila melanogaster function in a spatial and temporal regulatory hierarchy to control pattern formation during development. The precise spatial distribution of segmentation genes is an important aspect of their activity. Therefore, it has been the focus of this dissertation to analyze the spatio-temporal regulation of these genes during development. Three different classes of segmentation genes are examined. The role of the “pair-rule” class is examined in the regulation of the “segment polarity” class. Aspects of “homeotic” gene regulation are also analyzed. The first part of the thesis primarily deals with the regulation of the engrailed (en) segment polarity gene by the pair-rule genes. Use has been made of two different transgenes: (1) The pair-rule gene even-skipped (eve) under the control of the heat-shock promoter (hsp70). (2) The pair-rule gene runt (run) under the control of hsp70. Using these transgenes, various properties of the eve and run proteins and their role(s) in the process of en stripe regulation has been examined in embryos. As a result of these studies, a new model is proposed to explain the regulation of en stripes by the pair-rule genes. This model suggests that in contrast to the previous view, en is primarily regulated by negative and double negative mechanisms. The bithorax (bx) mutations in the Ultrabithorax (Ubx) cause homeotic transformations of anterior third thoracic structures (T3a) towards anterior second thoracic structures (T2a) in the adult. A corresponding loss of Ubx protein expression in T3a of bx imaginal discs accompanies these transformations. In the final part of this thesis, two genetic loci which modify the bx1-induced transformation are described. A locus which is mapped very close to the pink peach gene suppresses the bx1 phenotype. In contrast, mutations in the suppressor of sable gene enhance the bx 1 phenotype. A correlation was observed between patterns of Ubx protein expression and phenotypic transformations.Ph.D

Time- and Concentration-Dependent Response of the Drosophila Antenna Imaginal Disc to Antennapedia

AbstractThe response of the antenna imaginal disc to ectopic Antennapedia gene expression was explored in a heat shock Antennapedia (hsAntp) transgenic strain and in strains doubly transgenic for hsAntp and downstream enhancer trap targets, The distal to proximal changes in morphological transformation in response to Antennapedia product at different developmental stages were correlated with changing expression patterns of transgene targets from antenna to leg-like patterns. Dose-response studies indicated changing thresholds of response to Antennapedia. At particular stages and doses of Antennapedia product, cell differentiation of leg bristles was uncoupled from transformation of the third antennal segment to tarsus. The results suggest that determination for bristle type does not depend on a prior determination decision for organ type. The results also provide an avenue for exploring the nature of "competence" at cellular and molecular levels

A Role for the Segment Polarity Gene shaggy/GSK-3 in the Drosophila Circadian Clock

AbstractTissue-specific overexpression of the glycogen synthase kinase-3 (GSK-3) ortholog shaggy (sgg) shortens the period of the Drosophila circadian locomotor activity cycle. The short period phenotype was attributed to premature nuclear translocation of the PERIOD/TIMELESS heterodimer. Reducing SGG/GSK-3 activity lengthens period, demonstrating an intrinsic role for the kinase in circadian rhythmicity. Lowered sgg activity decreased TIMELESS phosphorylation, and it was found that GSK-3β specifically phosphorylates TIMELESS in vitro. Overexpression of sgg in vivo converts hypophosphorylated TIMELESS to a hyperphosphorylated protein whose electrophoretic mobility, and light and phosphatase sensitivity, are indistinguishable from the rhythmically produced hyperphosphorylated TIMELESS of wild-type flies. Our results indicate a role for SGG/GSK-3 in TIMELESS phosphorylation and in the regulated nuclear translocation of the PERIOD/TIMELESS heterodimer

The Porcupine Gene Is Required for Wingless Autoregulation in Drosophila

The Drosophila segment polarity gene wingless (wg) is required in the regulation of engrailed (en) expression and the determination of cell fates in neighboring cells. This paracrine wg activity also regulates transcription of wg itself, through a positive feedback loop including en activity. In addition, wg has a second, more direct autoregulatory requirement that is distinct from the en-dependent feedback loop. Four gene products, encoded by armadillo (arm), dishevelled (dsh), porcupine (porc) and zeste-white 3 (zw3), have been previously implicated as components of wg paracrine signaling. Here we have used three different assays to assess the requirements of these genes in the more direct wg autoregulatory pathway. While the activities of dsh, zw3 and arm appear to be specific to the paracrine feedback pathway, the more direct autoregulatory pathway requires porc.Version of Recor