Effects of protein kinase C modulation on NMDA receptor mediated regulation of neurotransmitter enzyme and c-fos protein in cultured neurons

The role of protein kinase C (PKC) in N-methyl-d-aspartate (NMDA) receptor-mediated biochemical differentiation and c-fos protein expression was investigated in cultured cerebellar granule neurons. The biochemical differentiation of glutamatergic granule cells was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 1 to 3 days (between 2 and 5 days in vitro), it elevated the specific activity of glutaminase. In contrast, NMDA had little effect on the activity of aspartate aminotransferase or of lactate dehydrogenase. Treatment of 10-day old granule neurons with NMDA also resulted in a marked increase in the immunocytochemically measured expression of c-fos protein. The increases in both the activity of glutaminase and the steady state level of c-fos protein were specific to the activation of NMDA receptors, as they were completely blocked byd,l-2-amino-5-phosphonovaleric acid. The specific stimulation of NMDA receptors in PKC-depleted granule neurons or in the presence of reasonably specific PKC inhibitors also produced significant elevation in the activity of glutaminase and the expression of c-fos protein. These increases were similar in magnitude to those observed in the granule neurons of the respective control groups. Our findings demonstrate that PKC is not directly involved in the NMDA receptor-mediated signal transduction processes associated with biochemical differentiation and c-fos induction in cerebellar granule neurons.     

Vertical stratification of beetles in tropical rainforests as sampled by light traps in North Queensland,Australia

How arthropods are distributed within the vertical structure of tropical rainforests is of considerable interest to ecologists. Here, we examine how light trapped beetles are distributed in tropical rainforest in North Queensland, Australia. In January and July 2012, traps were suspended 0 m, 10 m, 20 m and 30 m above the ground in five locations with no more than one trap at any single location on any night. Maximum canopy height at the sites was 35 m. A total of 7299 individuals of 492 morphospecies and 66 families were collected. The species abundance‐based coverage estimator predicted a total species richness of 765. Sample completeness decreases with increasing height from the ground suggesting higher strata were less well sampled. Distance‐based redundancy analysis showed species richness was significantly different between 30 m and all other levels but not between other paired strata. In contrast, both species composition and family composition were significantly distinct for all strata pairs except 10 m with 20 m, and 20 m with 30 m, suggesting that the most distinct strata were 0 m and 30 m. The first two axes of ordination and hierarchical clustering accounted for 46.5% and 17.4% of species composition variation corresponding with season and stratum, respectively. Family level analyses gave similar results to those at the species level. We found stratification of different feeding guilds with herbivores comprising a larger percentage of species in higher strata, whereas saprophages were restricted to the lower strata, reflecting the availability of key resources for these guilds. Fewer species or families were found to be indicators of strata, as measured using IndVal, than for Malaise and flight interception traps (FIT). Dytiscidae and Hydraenidae were abundant but had not been collected using Malaise and FIT. Which species or families are indicators of strata depends on sampling method suggesting multiple sampling methods should be used to establish indicators.     

Development of the peripheral glial cells in Drosophila

In complex organisms the nervous system comprises two cell types: neurons and glial cells. Their correct interplay is of crucial importance during both the development of the nervous system and for later function of the nervous system. In recent years tools have been developed for Drosophila that enable genetic approaches to understanding glial development and differentiation. Focusing on peripheral glial cells we first summarize wild-type development, then introduce some of the relevant genes that have been identified. Despite obvious differences between Drosophila and mammalian glial cells, the molecular machinery that controls terminal differentiation appears well conserved.     

Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy

Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.     

Expression of a ULK1/2 binding-deficient ATG13 variant can partially restore autophagic activity in ATG13-deficient cells

Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 has been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.     

The GAD65 knock out mouse – a model for GABAergic processes in fear‐ and stress‐induced psychopathology

The γ‐amino butyric acid (GABA) synthetic enzyme glutamic acid decarboxylase (GAD)65 is critically involved in the activity‐dependent regulation of GABAergic inhibition in the central nervous system. It is also required for the maturation of the GABAergic system during adolescence, a phase that is critical for the development of several neuropsychiatric diseases. Mice bearing a null mutation of the GAD65 gene develop hyperexcitability of the amygdala and hippocampus, and a phenotype of increased anxiety and pathological fear memory reminiscent of posttraumatic stress disorder. Although genetic association of GAD65 in human has not yet been reported, these findings are in line with observations of reduced GABAergic function in these brain regions of anxiety disorder patients. The particular value of GAD65(?/?) mice thus lies in modeling the effects of reduced GABAergic function in the mature nervous system. The expression of GAD65 and a second GAD isozyme, GAD67, are differentially regulated in response to stress in limbic brain areas suggesting that by controlling GABAergic inhibition these enzymes determine the vulnerability for the development of pathological anxiety and other stress‐induced phenotypes. In fact, we could recently show that GAD65 haplodeficiency, which results in delayed postnatal increase of GABA levels, provides resilience to juvenile‐stress‐induced anxiety to GAD65(+/?) mice thus foiling the increased fear and anxiety in homozygous GAD65(?/?) mice.