The atypical cadherin fat directly regulates mitochondrial function and metabolic state.

Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. Here, we show Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ft(mito)) that is imported into mitochondria. Ft(mito) binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect.

Functional interactions between Fat family cadherins in tissue morphogenesis and planar polarity.

The atypical cadherin fat (ft) was originally discovered as a tumor suppressor in Drosophila and later shown to regulate a form of tissue patterning known as planar polarity. In mammals, four ft homologs have been identified (Fat1-4). Recently, we demonstrated that Fat4 plays a role in vertebrate planar polarity. Fat4 has the highest homology to ft, whereas other Fat family members are homologous to the second ft-like gene, ft2. Genetic studies in flies and mice imply significant functional differences between the two groups of Fat cadherins. Here, we demonstrate that Fat family proteins act both synergistically and antagonistically to influence multiple aspects of tissue morphogenesis. We find that Fat1 and Fat4 cooperate during mouse development to control renal tubular elongation, cochlear extension, cranial neural tube formation and patterning of outer hair cells in the cochlea. Similarly, Fat3 and Fat4 synergize to drive vertebral arch fusion at the dorsal midline during caudal vertebra morphogenesis. We provide evidence that these effects depend on conserved interactions with planar polarity signaling components. In flies, the transcriptional co-repressor Atrophin (Atro) physically interacts with Ft and acts as a component of Fat signaling for planar polarity. We find that the mammalian orthologs of atro, Atn1 and Atn2l, modulate Fat4 activity during vertebral arch fusion and renal tubular elongation, respectively. Moreover, Fat4 morphogenetic defects are enhanced by mutations in Vangl2, a 'core' planar cell polarity gene. These studies highlight the wide range and complexity of Fat activities and suggest that a Fat-Atrophin interaction is a conserved element of planar polarity signaling.

Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells.

Cortical spreading depression (CSD) induces waves of neuronal depolarization that confer neuroprotection to subsequent ischemic events in the rat brain. To gain insights into the molecular mechanisms elicited by CSD, we used representational difference analysis (RDA) to identify mRNAs induced by potassium depolarization in vivo. Using this approach, we have isolated a cDNA encoding the SIM2-related bHLH-PAS protein Nxf. Our results confirm that Nxf mRNA and protein are rapidly and transiently expressed in cortical neurons following CSD. Reporter assays show that Nxf is a transcriptional activator that associates with the bHLH-PAS sub-class co-factor ARNT2. Adenovirus-mediated expression of epitope-tagged Nxf results in cell death and the direct activation of the Bax gene in cultured cells. However, RNA interference studies show that endogenous Nxf is required for optimal neuroprotection by preconditioning in cultured F-11 cells. Together, our data indicate that Nxf is a novel bHLH-PAS transactivator transiently induced by preconditioning and that its sustained expression is detrimental. The identification of Nxf may represent an important step in our understanding of the molecular mechanisms of brain preconditioning and injury.

The benzodiazepine diazepam demonstrates the usefulness of Syrian hamsters as a model for anxiety testing: evaluation of other classes of anxiolytics in comparison to diazepam.

Clinical evidence in humans suggests that there is some linkage between dysfunction in the timing of circadian rhythms and certain types of depression. In animal models, Syrian hamsters have been used extensively to study the pharmacology of circadian rhythms, while rats and mice are used to screen putative anxiolytics/antidepressant compounds. It would be beneficial to be able to test anxiolytic/antidepressant compounds in hamsters in conjunction with circadian rhythm studies. Therefore, in this study, Syrian hamsters were used in three experimental paradigms to evaluate anxiety: the elevated plus maze, the t-tube, and the open field Thatcher-Britton conflict test. Diazepam, tested with 2mg/kg and 5mg/kg intraperitoneal injections, was found to induce anxiolytic activity in each of the three tests. Hamsters were more likely to spend time in the open arms in the plus maze, displayed more exploratory behavior in the t-tube, and were quicker to enter a brightly lit exposed field in the Thatcher-Britton conflict test following injections of diazepam. Diazepam (2mg/kg) was also tested at three times during the 24-h day in the elevated plus maze: at the beginning and end of the lights-on period (Zeitgeber times 23 and 11, respectively) and once in the dark just before the room lights came on (Zeitgeber time 20). Diazepam induced anxiolytic activity only at Zeitgeber 23. Therefore, the following known and putative anxiolytic compounds were also evaluated in each of the three tests at Zeitgeber 23: citalopram, the neurokinin(1) receptor antagonists GR205171 and vestipitant, the corticotropin releasing factor(1) receptor antagonist CP154526, the cannabinoid receptor(1) agonist CP55940, the serotonin(6) receptor antagonist SB399885, and the metabotropic glutamate receptor(5) antagonists fenobam and MTEP. Vestipitant displayed some anxiolytic activity in the elevated plus maze, but this effect was not confirmed with GR205171. None of the other compounds displayed any anxiolytic activity in the tests. Nevertheless, the present results with diazepam - together with a few reports from other laboratories, indicate that the elevated plus maze may be a suitable procedure for evaluating the actions of anxiolytic compounds in Syrian hamsters. In view of current interest in novel classes of psychotropic agent interacting with diverse GABA(A) receptor recognition sites, further characterization appears justified.

Loss of Fat4 disrupts PCP signaling and oriented cell division and leads to cystic kidney disease.

Tissue organization in Drosophila is regulated by the core planar cell polarity (PCP) proteins Frizzled, Dishevelled, Prickle, Van Gogh and Flamingo. Core PCP proteins are conserved in mammals and function in mammalian tissue organization. Recent studies have identified another group of Drosophila PCP proteins, consisting of the protocadherins Fat and Dachsous (Ds) and the transmembrane protein Four-jointed (Fj). In Drosophila, Fat represses fj transcription, and Ds represses Fat activity in PCP. Here we show that Fat4 is an essential gene that has a key role in vertebrate PCP. Loss of Fat4 disrupts oriented cell divisions and tubule elongation during kidney development, leading to cystic kidney disease. Fat4 genetically interacts with the PCP genes Vangl2 and Fjx1 in cyst formation. In addition, Fat4 represses Fjx1 expression, indicating that Fat signaling is conserved. Together, these data suggest that Fat4 regulates vertebrate PCP and that loss of PCP signaling may underlie some cystic diseases in humans.