Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes.

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

Differences in phosphorylation of human and chicken stathmin by MAP kinase.

Stathmin/Op18 is a highly conserved 19 kDa cytosolic phosphoprotein. Human and chicken stathmin share 93% identity with only 11 amino acid substitutions. One of the substituted amino acids is serine 25, which is a glycine in chicken stathmin. In human stathmin, serine 25 is the main phosphorylation site for MAP kinase. In this study, we have compared the phosphorylation of human and chicken stathmin. The proteins were expressed in Sf9 cells using the baculovirus expression system and purified for in vitro phosphorylation assays. Phosphorylation with MAP kinase showed that chicken stathmin was phosphorylated 10 times less than human stathmin. To identify the phosphorylation sites we used liquid chromatography/mass spectrometry (LC/MS/MS). The only amino acid found phosphorylated was serine 38, which corresponds to the minor phosphorylation site in human stathmin. Phosphorylation with p34(cdc2)- and cGMP-dependent protein kinases gave almost identical phosphorylation levels in the two stathmins.

Localization and targeting of SCG10 to the trans-Golgi apparatus and growth cone vesicles.

SCG10 is a membrane-associated, microtubule-destabilizing protein of neuronal growth cones. Using immunoelectron microscopy, we show that in the developing cortex of mice, SCG10 is specifically localized to the trans face Golgi complex and apparently associated with vesicular structures in putative growth cones. Consistent with this, subcellular fractionation of rat forebrain extracts demonstrates that the protein is enriched in the fractions containing the Golgi apparatus and growth cone particles. In isolated growth cone particles, SCG10 was found to be particularly concentrated in the growth cone vesicle fraction. To evaluate the molecular determinants of the specific targeting of SCG10 to growth cones, we have transfected PC12 cells and primary neurons in culture with mutant and fusion cDNA constructs. Deletion of the amino-terminal domain or mutations within this domain that prevented palmitoylation at cysteines 22 and 24 abolished Golgi localization as well as growth cone targeting, suggesting that palmitoylation of the amino-terminal domain is a necessary signal for Golgi sorting and possibly transport of SCG10 to growth cones. Fusion proteins consisting of the amino-terminal domain of SCG10 and the cytosolic proteins stathmin or glutathione-S-transferase colocalized with a Golgi marker, alpha-mannosidase II, and accumulated in growth cones of both axons and dendrites. These results reveal a novel axonal/dendritic growth cone targeting sequence that involves palmitoylation.