Phosphatidylinositol-5-Phosphate 4-Kinases Regulate Cellular Lipid Metabolism By Facilitating Autophagy.

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P2) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P2, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a-/-Pip4k2b-/- mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P2 synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.

Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers.

Cancer cells reprogram their metabolism to promote growth and proliferation. The genetic evidence pointing to the importance of the amino acid serine in tumorigenesis is striking. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of serine biosynthesis, is overexpressed in tumors and cancer cell lines via focal amplification and nuclear factor erythroid-2-related factor 2 (NRF2)-mediated up-regulation. PHGDH-overexpressing cells are exquisitely sensitive to genetic ablation of the pathway. Here, we report the discovery of a selective small molecule inhibitor of PHGDH, CBR-5884, identified by screening a library of 800,000 drug-like compounds. CBR-5884 inhibited de novo serine synthesis in cancer cells and was selectively toxic to cancer cell lines with high serine biosynthetic activity. Biochemical characterization of the inhibitor revealed that it was a noncompetitive inhibitor that showed a time-dependent onset of inhibition and disrupted the oligomerization state of PHGDH. The identification of a small molecule inhibitor of PHGDH not only enables thorough preclinical evaluation of PHGDH as a target in cancers, but also provides a tool with which to study serine metabolism.

Hippo and PI5P4K signaling intersect to control the transcriptional activation of YAP.

Phosphoinositides are essential signaling molecules. The PI5P4K family of phosphoinositide kinases and their substrates and products, PI5P and PI4,5P2, respectively, are emerging as intracellular metabolic and stress sensors. We performed an unbiased screen to investigate the signals that these kinases relay and the specific upstream regulators controlling this signaling node. We found that the core Hippo pathway kinases MST1/2 phosphorylated PI5P4Ks and inhibited their signaling in vitro and in cells. We further showed that PI5P4K activity regulated several Hippo- and YAP-related phenotypes, specifically decreasing the interaction between the key Hippo proteins MOB1 and LATS and stimulating the YAP-mediated genetic program governing epithelial-to-mesenchymal transition. Mechanistically, we showed that PI5P interacted with MOB1 and enhanced its interaction with LATS, thereby providing a signaling connection between the Hippo pathway and PI5P4Ks. These findings reveal how these two important evolutionarily conserved signaling pathways are integrated to regulate metazoan development and human disease.

Age-dependent and strain-dependent influences of morphine on mouse social investigation behavior.

Opioid-coded neural circuits play a substantial role in how individuals respond to drugs of abuse. Most individuals begin using such drugs during adolescence and within a social context. Several studies indicate that adolescent mice exhibit a heightened sensitivity to the effects of morphine, a prototypical opiate drug, but it is unclear whether these developmental differences are related to aspects of motivated behavior. Moreover, exposure to opioids within the rodent brain can alter the expression of social behavior, yet little is known about whether this relationship changes as a function of development or genetic variation. In this study, we conducted a series of experiments to characterize the relationship between genetic background, adolescent development and morphine-induced changes in mouse social investigation (SI). At two time points during adolescent development [postnatal days (PD) 25 and 45], social interactions of test mice of the gregarious C57BL/6J (B6) strain were more tolerant to the suppressive effects of morphine [effective dose 50 (ED50)=0.97 mg/kg and 2.17 mg/kg morphine, respectively] than test mice from the less social BALB/cJ (BALB) strain (ED50=0.61 mg/kg and 0.91 mg/kg morphine, respectively). By contrast, this strain-dependent difference was not evident among adult mice on PD 90 (ED50=1.07 mg/kg and 1.41 mg/kg morphine for BALB and B6 mice, respectively). An additional experiment showed that the ability of morphine to alter social responsiveness was not directly related to drug-induced changes in locomotor behavior. Finally, administration of morphine to stimulus mice on PD 25 reduced social investigation of test mice only when individuals were from the B6 genetic background. Overall, these results indicate that alterations in endogenous opioid systems are related to changes in SI that occur during adolescence, and that morphine administration may mimic rewarding aspects of social encounter.

Differential entrainment of a social rhythm in adolescent mice.

Daily routines in animal activities range from sleep-wake cycles, to foraging bouts, to social interactions. Among animals living within groups, it is unclear whether the motivations that underlie social interactions respond to daily light-dark (LD) cycles or endogenous circadian rhythms. Employing two mouse strains (BALB/cJ [BALB] and C57BL/6J [B6]) with genetically based differences in social affect and circadian rhythms, we examined how social investigation (SI) is modulated by social deprivation and circadian factors. We found a genetic influence on SI that was moderated by the preceding duration of social deprivation, requiring 3-6 h of social isolation prior to testing. Following 6h of social deprivation, the SI responses of adolescent B6 mice were greater than those of BALB mice only when the isolation period was imposed during the dark phase of the LD cycle. When B6 mice were weaned into conditions of constant darkness, a novel, endogenous social rhythm emerged, which was characterized by two pronounced peaks of social responsiveness (relative to one peak under LD entrainment) that were separated by 12-h intervals. Irrespective of the lighting conditions during social isolation, the SI responses of adolescent BALB mice did not oscillate across the day. Similar strain-dependent patterns of sociability were evident within groups of mice that were left undisturbed in their home cage under LD entrainment or constant darkness. Overall, genetic influences on the social phenotypes of adolescent mice are thus moderated by an interaction between social deprivation and oscillations of an endogenous social rhythm that entrains to the LD cycle.