Striatal expression of a calmodulin fragment improved motor function, weight loss, and neuropathology in the R6/2 mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, caused by a polyglutamine expansion in the huntingtin protein (htt). Increasing evidence suggests that transglutaminase (TGase) plays a critical role in the pathophysiology of HD possibly by stabilizing monomeric, polymeric and aggregated htt. We previously reported that in HEK293 and SH-SY5Y cells expression of a calmodulin (CaM)-fragment, consisting of amino acids 76-121 of CaM, decreased binding of CaM to mutant htt, TGase-modified htt and cytotoxicity associated with mutant htt and normalized intracellular calcium release. In this study, an adeno-associated virus (AAV) that expresses the CaM-fragment was injected into the striatum of HD transgenic R6/2 mice. The CaM-fragment significantly reduced body weight loss and improved motor function as indicated by improved rotarod performance, longer stride length, lower stride frequency, fewer low mobility bouts and longer travel distance than HD controls. A small but insignificant increase in survival was observed in R6/2 mice with CaM-fragment expression. Immunoprecipitation studies show that expression of the CaM-fragment reduced TGase-modified htt in the striatum of R6/2 mice. The percentage of htt-positive nuclei and the size of intranuclear htt aggregates were reduced by the CaM-fragment without striatal volume changes. The effects of CaM-fragment appear to be selective, as activity of another CaM-dependent enzyme, CaM-dependent kinase II, was not altered. Moreover, inhibition of TGase-modified htt was substrate-specific since overall TGase activity in the striatum was not altered by treatment with the CaM-fragment. Together, these results suggest that disrupting CaM-htt interaction may provide a new therapeutic strategy for HD.

Protective effects of interrupting the binding of calmodulin to mutant huntingtin.

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.

Mutant huntingtin protein: a substrate for transglutaminase 1, 2, and 3.

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.

Phospholipase C, Ca2+, and calmodulin signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by transglutaminase.

RATIONALE: Serotonin and especially serotonin 2A (5-HT(2A)) receptor signaling are important in the etiology and treatment of schizophrenia and affective disorders. We previously reported a novel 5-HT(2A) receptor effector, increased transglutaminase (TGase)-catalyzed transamidation, and activation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. OBJECTIVES: In this study, we explore the signaling pathway involved in 5-HT(2A) receptor-mediated Rac1 transamidation. METHODS: A1A1v cells were pretreated with pharmacological inhibitors of phospholipase C (PLC) or calmodulin (CaM), and then stimulated by the 5-HT(2A) receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). Intracellular Ca(2+) concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca(2+) by a Ca(2+) ionophore or a chelating agent on Rac1 transamidation was also evaluated. RESULTS: In cells pretreated with a PLC inhibitor U73122, DOI-stimulated increases in the intracellular Ca(2+) concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca(2+) chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca(2+) ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca(2+) to a level comparable to cells treated with DOI, produced an increase in TGase-modified Rac1 without 5-HT(2A) receptor activation. Moreover, the CaM inhibitor W-7, significantly decreased Rac1 transamidation in a dose-dependent manner in DOI-treated cells. CONCLUSIONS: These results indicate that 5-HT(2A) receptor-coupled PLC activation and subsequent Ca(2+) and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca(2+) is sufficient to induce Rac1 transamidation.

Neuroprotective effects of calmodulin peptide 76-121aa: disruption of calmodulin binding to mutant huntingtin.

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 microM 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.

Phenotypic deconstruction reveals involvement of manganese transporter malvolio in honey bee division of labor.

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.