Transglutaminase (TGase), nature’s biological glue, catalyzes the post-translational modification of proteins by formation of intra- and intermolecular protein cross-links or by primary amine incorporation. TGase has various physiological functions, such as skin-barrier formation and blood clot stabilization, whereas increasing evidence indicates they may also involved in neurodegenerative diseases including Huntington’s disease (HD), Alzheimer disease’s, and progressive supranuclear palsy. Mutant huntingtin (htt) and small G proteins (e.g. Rac 1) are potential substrates of TGases. The purpose of this dissertation was to characterize the mechanisms by which 5-HT2A receptor signaling and calmodulin (CaM) regulate TGase-catalyzed transamidation of Rac1 and htt in cultured neuronal cells and HD transgenic animals, respectively.
5-HT2A receptors are G-protein coupled receptors which are widely expressed in the brain, peripheral vasculature, platelets and skeletal muscle. They are involved in diverse physiological functions, from platelet aggregation to neuroendocrine release. In A1A1v cells, a rat cortical cell line, stimulation of 5-HT2A receptor induces small G protein Rac1 transamidation and activation. An inhibitory agent or knockdown of TGase by siRNA revealed that TGase is responsible for transamidation and activation of Rac1. Moreover, serotonin was identified as an amine that becomes transamidated to Rac1 by TGase. The classical signal transduction pathway of 5-HT2A receptors is Gq/11-coupled activation of phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacyglycerol. IP3 mobilizes Ca2+ from the endoplasmic reticulum and thereby increases intracellular Ca2+ and consequently, it may enhance TGases enzymatic activity. Inhibition of PLC or manipulation of intracellular Ca2+ by a chelating agent suppressed 5-HT 2A receptor-mediated Rac1 transamidation, whereas an elevation in intracellular Ca2+ via an ionophore can mimic 5-HT2A receptor-induced cytosolic Ca2+ increases and is sufficient to induce TGase-catalyzed Rac1 transamidation. Moreover, a CaM inhibitor decreased 5-HT 2A receptor-stimulated Rac1 modification by TGase in a dose-dependent manner. These results suggest that PLC, Ca2+ and CaM signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by TGase.
In the next study, we propose that interrupting CaM interactions with htt is therapeutically beneficial in HD. This hypothesis was based on the observations that CaM regulates TGase-modification of mutant htt in cells and co-localizes with TGase and htt in intranuclear inclusions in HD cortex. Furthermore the association of CaM with mutant htt was demonstrated by affinity purification and coimmunoprecipitation approaches. Our previous studies demonstrated that in HEK293 and SH-SY5Y cells, expression of a CaM-fragment, consisting of amino acids 76-121 of CaM, decreased binding of CaM to mutant htt, decreased TGase-modified htt, decreased 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 and littermate control mice. The HD mice with CaM-fragment expression had significantly reduced body weight loss and improved motor function compare to HD control mice. Without affecting the activity of CaM-dependent enzymes such as CaM-dependent kinase II, CaM-fragment specifically reduced TGase-modified htt, the percentage of htt-positive nuclei and the size of intranuclear htt aggregates in HD mouse striatum. Thus, disrupting CaM-htt interaction with CaM-fragment may provide a new therapeutic strategy for HD patients.
The data presented here support our hypothesis that stimulation of 5-HT 2A receptors induces TGase-catalyzed Rac1 transamidation and activation by PLC and Ca2+/CaM signaling, whereas disruption of CaM-htt interaction inhibits TGase-catalyzed modification of htt and provides neuronal protection in HD. These results further suggest that CaM regulates the TGase transamidation reaction and TGase modification of htt is involved in the formation and stabilization of htt-aggregates and may play a role in the pathogenesis of HD.