Essential roles of class E Vps proteins for sorting into multivesicular bodies in Schizosaccharomyces pombe

The multivesicular body (MVB) sorting pathway is required for a number of biological processes, including downregulation of cell-surface proteins and protein sorting into the vacuolar lumen. The function of this pathway requires endosomal sorting complexes required for transport (ESCRT) composed of class E vacuolar protein sorting (Vps) proteins in Saccharomyces cerevisiae, many of which are conserved in Schizosaccharomyces pombe. Of these, sst4/vps27 (homologous to VPS27) and sst6 (similar to VPS23) have been identified as suppressors of sterility in ste12Δ (sst), although their functions have not been uncovered to date. In this report, these two sst genes are shown to be required for vacuolar sorting of carboxypeptidase Y (CPY) and an MVB marker, the ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein, despite the lack of the ubiquitin E2 variant domain in Sst6p. Disruption mutants of a variety of other class E vps homologues also had defects in sorting of CPY and Ub–GFP–CPS. Sch. pombe has a mammalian AMSH homologue, sst2. Phenotypic analyses suggested that Sst2p is a class E Vps protein. Taken together, these results suggest that sorting into multivesicular bodies is dependent on class E Vps proteins, including Sst2p, in Sch. pombe

Metabolic mapping of rat brain activity associated with conditioned fear extinction and renewal, and improvement of extinction memory by the metabolic enhancer methylene blue

textChanges in brain metabolism associated with the consolidation, extinction and recall of fear memories were investigated in rats using two complementary brain metabolic mapping approaches. First, fluorodeoxyglucose (FDG) metabolic mapping technique was used to track the stimulus-evoked changes in brain glucose uptake that mostly occur during the first ten minutes following the FDG administration. Second, cytochrome oxidase (CO) histochemistry, which is wellsuited for tracking long-term changes in brain metabolic capacity, was utilized. By combining these two techniques, brain structures involved in fear extinction memory consolidation and retention were compared to brain regions that displayed altered metabolic activity during conditioned fear memory recall. Additionally, since memory consolidation requires expenditure of energy, enhancement of brain oxidative phosphorylation through CO activity increase was tested as a possible way for improving extinction memory retention in rats. Low doses of the metabolic enhancer methylene blue (MB) were used to enhance CO activity in the post-extinction training period, to test the hypothesis that neurons with high metabolic demand which are engaged in consolidation and retention of the extinction memory would benefit most from the presence of a metabolic-enhancing drug. The results suggest that during conditioned fear renewal, the auditory conditioned stimulus activates the neural representation of the footshock unconditioned stimulus, thus supporting Pavlov’s stimulussubstitution model of classical conditioning. Quantitative CO histochemistry revealed that Pavlovian fear acquisition training increased metabolic capacity in several brain regions, including medial prefrontal cortex (mPFC) and septum, while extinction training reduced CO activity to levels comparable to the pseudorandom group. A functional neural network model of extinction explored how the direct influences on regions such as mPFC and amygdala might change between fear extinction recall and fear renewal. Finally, the third experiment illustrated that MB might be a useful adjunct to exposure therapy, since it improved consolidation and retention of fear extinction in our animal model of specific phobias.Institute for Neuroscienc

Atomic Structure, Electronic Properties, and Reactivity of In-Plane Heterostructures of Graphene and Hexagonal Boron Nitride

We applied density functional theory (DFT) to investigate structural and electronic properties, as well as the reactivity of in-plane heterostructures composed of graphene and hexagonal boron nitride (h-BN). The calculations demonstrate a strong tendency of graphene and h-BN to minimize the number of C–N and C–B bonds and thus to segregate into homogeneous domains. A simple bond model, with parameters obtained from DFT calculations, is used to describe trends in the formation energies of the studied heterostructures. We show that the electronic properties of the BN clusters embedded into graphene qualitatively resemble those of graphene antidot lattices. The calculations also reveal that the h-BN monolayer doped with small graphene clusters is a material with the band gap tunable over an energy range of several electron volts, since the band gap values strongly depend on the size of embedded graphene quantum dots. The reactivity of the graphene/h-BN heterostructures is quantified using H atoms as a probe. We found a strong increase of the H binding energy in the heterostructures, where localized electronic states appear in the vicinity of the Fermi level. The highest value of 2.31 eV, calculated for the ideal zigzag graphene/h-BN interface, is approximately three times larger compared to the H atom binding energy at an infinite graphene sheet

The Calcium Oscillator of GnRH-1 Neurons Is Developmentally Regulated

Oscillations in intracellular calcium levels have been described in GnRH-1 neurons in both prenatal and adult cells. However, differences have been reported in the mechanisms underlying these [Ca2+]i oscillations, dependent on the model used. The goal of this study was to address whether these changes depend on the maturation status of GnRH-1 neurons by assaying prenatal GnRH-1 cells maintained in explants, at two different developmental stages. This report documents an increase in the frequency of [Ca2+]i oscillations between 1 and 3 wk of in vitro maturation. During the early stage, [Ca2+]i oscillations are blocked by tetrodotoxin and are mainly triggered by excitatory neurotransmitters, γ-aminobutyric acid (GABA), and glutamate. In contrast, in the later stage, some cells exhibit residual tetrodotoxin-insensitive [Ca2+]i oscillations, which are sustained by action potential-independent GABA and glutamate release. The strength of these two excitatory inputs remained relatively constant during the maturation process, and the increase in frequency of [Ca2+]i oscillations observed at the later stage is due to a novel excitatory input carried by cholecystokinin. Together, these data indicate developmentally regulated release and interactions of neurotransmitters (known regulators of GnRH-1 cells in adults) and point to extrinsic factors regulating GnRH-1 cellular physiology

Decreased Expression of A-Kinase Anchoring Protein 150 in GT1 Neurons Decreases Neuron Excitability and Frequency of Intrinsic Gonadotropin-Releasing Hormone Pulses

The frequency of intrinsic pulsatile GnRH secretion from endogenous GnRH neurons and GT1 GnRH cell lines is stimulated by increased intracellular cAMP levels. The downstream molecules comprising the cAMP signaling pathway are organized in microdomains by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). These molecules tether protein kinase A, cAMP-specific phosphodiesterases, phosphatases to known substrates. In neurons AKAP150 organizes many of the signaling molecules known to regulate the excitability and intrinsic pulsatile activity of GnRH neurons. AKAP150 was expressed in both the GT1-1 and GT1-7 cells. We determined the role of AKAP150 in coordinating GT1-1 cell excitability and intrinsic GnRH pulsatile secretion by lowering AKAP150 levels with a small interfering RNA (siRNA) adenovirus construct to AKAP150 (Ad-AKAP150-siRNA). Infection with Ad-AKAP150-siRNA specifically decreased AKAP150 mRNA levels by 74% and protein levels by 53% relative to uninfected cells or cells infected with a luciferase control adenovirus siRNA vector. In GT1 cells, spontaneous Ca2+ oscillations, an index of neuron excitability, are stimulated by increased levels of intracellular cAMP and lowered by decreased levels. The frequency of spontaneous Ca2+ oscillations in Ad-AKAP150-siRNA-treated GT1-1 cells decreased by 47.2% relative to controls. A dramatic decrease in the number of spontaneous GnRH pulses was also observed after infection with Ad-AKAP150-siRNA. The interpulse interval increased to 143 ± 20.25 min in Ad-AKAP150-siRNA infected cells from 32.2 ± 7.3 min in luciferase control adenovirus siRNA vector-infected cells. These data demonstrate an important role of AKAP150 in coordinating signaling events regulating the frequency of intrinsic pulsatile GnRH secretion