Molecular analysis of a complex behavioral phenotype is facilitated by dissecting it into simpler behavioral components. Using this approach, we present evidence implicating increased manganese transport by the malvolio (mvl) gene into brain cells as one factor that influences age-related division of labor in honey bee colonies. We studied mvl because manganese affects sucrose responsiveness in Drosophila melanogaster, and sucrose responsiveness is related to division of labor in honey bee colonies. Honey bee foragers are more responsive to sucrose in the laboratory than are younger nurse bees, and pollen foragers are more responsive to sucrose than nectar foragers. Levels of mvl mRNA in the brain and manganese in the head were higher in pollen foragers compared with nurses, with nectar foragers intermediate. Manganese treatment increased honey bee sucrose responsiveness and caused precocious foraging. Manganese levels showed a similar pattern to mvl mRNA but manganese treatment did not increase pollen foraging. These results suggest that, while there are molecular pathways common to sucrose responsiveness and division of labor, linkages between a complex behavior and some of its simpler behavioral components are not obligatory. Together with previous findings, these results support the idea that some feeding-related genes in Drosophila have been used in social evolution to regulate division of labor.

Transglutaminase (TGase)-induced activation of small G proteins via 5-hydroxytryptamine (HT) 2A receptor signaling leads to platelet aggregation (Cell 115:851-862, 2003). We hypothesize that stimulation of 5-HT 2A receptors in neurons activates TGase, resulting in transamidation of serotonin to a small G protein, Rac1, thereby constitutively activating Rac1. Using immunoprecipitation and immunoblotting, we show that, in rat cortical cell line A1A1v, serotonin increases TGase-catalyzed transamidation of Rac1. This transamidation occurs in both undifferentiated and differentiated cells. Treatment with a 5-HT 2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine, but not the 5-HT 1A receptor agonist 5-hydroxy-2-dipropylamino tetralin, increases transamidation of Rac1 by TGase. In A1A1v cells, 5-HT 2A receptors mediate the transamidation reaction because expression of 5-HT 2C receptors was not detectable and the selective 5-HT 2A receptor antagonist blocked transamidation. Time course studies demonstrate that transamidation of Rac1 is significantly elevated after 5 and 15 min of serotonin treatment, but returns it to control levels after 30 min. The activity of Rac1 is also transiently increased following serotonin stimulation. Inhibition of TGase by cystamine or small interfering RNA reduces TGase modification of Rac1, and cystamine also prevents Rac1 activation. Serotonin itself is bound to Rac1 by TGase following 5-HT 2A receptor stimulation as demonstrated by coimmunoprecipitation experiments and a dose-dependent decrease of serotonin-associated Rac1 by cystamine. These data support the hypothesis that Rac1 activity is transiently increased due to TGase-catalyzed transamidation of serotonin to Rac1 via stimulation of 5-HT 2A receptors. Activation of Rac1 via TGase is a novel effector and second messenger of the 5-HT 2A receptorsignaling cascade in neurons.

Huntington's disease (HD) is a neurodegenerative disease caused by mutant huntingtin protein containing an expanded polyglutamine tract, which may cause abnormal protein–protein interactions such as increased association with calmodulin (CaM). We previously demonstrated in HEK293 cells that a peptide containing amino acids 76-121 of CaM (CaM-peptide) interrupted the interaction between CaM and mutant huntingtin, reduced mutant huntingtin-induced cytotoxicity and reduced transglutaminase (TG)-modified mutant huntingtin. We now report that adeno-associated virus (AAV)-mediated expression of CaM-peptide in differentiated neuroblastoma SH-SY5Y cells, stably expressing an N-terminal fragment of huntingtin containing 148 glutamine repeats, significantly decreases the amount of TG-modified huntingtin and attenuates cytotoxicity. Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II. In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 µM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein. These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

There is evidence suggesting that transglutaminase (TG) 2 plays a role in stabilizing monomeric and aggregated huntingtin, thereby contributing to the pathophysiology of Huntington disease. Calmodulin (CaM) regulates TG2 cross-linking of N-terminal mutant huntingtin in cells and colocalizes with TG and huntingtin in inclusions in Huntington disease cortex. The current study examined the effects of small fragments of CaM in human embryonic kidney 293T cells expressing N-terminal mutant huntingtin and transglutaminase 2. Four CaM fragments were developed: first 76 amino acids, last 72 amino acids, 77 amino acids in the center (CaM-center), and the overlapping region of last 72 amino acids and CaM-center (CaM-overlap). The last 72 amino acids, CaM-center, and CaM-overlap significantly decreased amounts of TG-modified huntingtin by 40% to 60%, and cytotoxicity decreased up to 40% compared with cells not expressing any CaM construct. Carbachol-stimulated release of intracellular calcium is significantly higher in cells expressing N-terminal mutant huntingtin and TG2 compared with vector-transfected cells; expression of either CaM-center or CaM-overlap in these cells returned the levels of carbachol-stimulated intracellular calcium release to control values. Furthermore, CaM-overlap expression significantly decreased huntingtin binding to CaM. These data further suggest that CaM regulates TG2 activity, plays a role in the disease-related modifications to mutant huntingtin, and that disruption of CaM-huntingtin interaction is potentially a new target for therapeutic intervention in Huntington disease.

The most prominent neuropathologic hallmarks of Huntington disease (HD) are cortical and striatal perinuclear cytoplasmic aggregates and intranuclear inclusions of mutant huntingtin. Our laboratory previously demonstrated that huntingtin protein colocalizes with transglutaminase 2 and its product, the epsilon-(gamma-glutamyl)lysine bond in intranuclear inclusions in HD frontal cortex. We also found that transglutaminase 2 cross-links N-terminal fragments of mutant huntingtin (htt-N63-148Q-myc) in cells in culture. We now report a significant increase in transglutaminase 2 mRNA in HD cortex (225% of controls) and striatum (399% of controls). Expression of the short transglutaminase 2 mRNA splice variant was not detectable in HD, although previous studies demonstrated upregulation in Alzheimer disease and progressive supranuclear palsy. Cells co-transfected with GFP-tagged transglutaminase 1, 2, or 3 and htt-N63-148Q-myc exhibit increased cross-linked huntingtin in the insoluble fraction of cell lysates. Treatment of cells with cystamine, a chemical inhibitor of transglutaminase, decreased aggregated and cross-linked huntingtin and increased viability of cells that were transfected with transglutaminase 2 and htt-N63-148Q-myc. These data suggest that transglutaminase 1, 2, and 3 could be involved in cross-linking of huntingtin into intranuclear inclusions in HD and that inhibiting transglutaminase should be explored as a potential treatment strategy for HD.