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 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

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-HT2A) receptor signaling are important in the etiology and treatment of schizophrenia and affective disorders. We previously reported a novel 5-HT2A 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-HT2A 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-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). Intracellular Ca2+ concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca2+ by a Ca2+ 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 Ca2+ concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca2+ chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca2+ ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca2+ to a level comparable to cells treated with DOI, produced an increase in TGase-modified Rac1 without 5-HT2A 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-HT2A receptorcoupled PLC activation and subsequent Ca2+ and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca2+ is sufficient to induce Rac1 transamidation

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

This is the published version, also available here: http://dx.doi.org/10.1523/JNEUROSCI.3307-09.2009.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

Dramatic age-related changes in nuclear and genome copy number in the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has become one of the most widely used model systems for the study of aging, yet very little is known about how C. elegans age. The development of the worm, from egg to young adult has been completely mapped at the cellular level, but such detailed studies have not been extended throughout the adult lifespan. Numerous single gene mutations, drug treatments and environmental manipulations have been found to extend worm lifespan. To interpret the mechanism of action of such aging interventions, studies to characterize normal worm aging, similar to those used to study worm development are necessary. We have used 4′,6′-diamidino-2-phenylindole hydrochloride staining and quantitative polymerase chain reaction to investigate the integrity of nuclei and quantify the nuclear genome copy number of C. elegans with age. We report both systematic loss of nuclei or nuclear DNA, as well as dramatic age-related changes in nuclear genome copy number. These changes are delayed or attenuated in long-lived daf-2 mutants. We propose that these changes are important pathobiological characteristics of aging nematodes

Cyberbiosecurity: A New Perspective on Protecting U.S. Food and Agricultural System

Our national data and infrastructure security issues affecting the “bioeconomy” are evolving rapidly. Simultaneously, the conversation about cyber security of the U.S. food and agricultural system (cyber biosecurity) is incomplete and disjointed. The food and agricultural production sectors influence over 20% of the nation's economy ($6.7T) and 15% of U.S. employment (43.3M jobs). The food and agricultural sectors are immensely diverse and they require advanced technologies and efficiencies that rely on computer technologies, big data, cloud-based data storage, and internet accessibility. There is a critical need to safeguard the cyber biosecurity of our bio economy, but currently protections are minimal and do not broadly exist across the food and agricultural system. Using the food safety management Hazard Analysis Critical Control Point system concept as an introductory point of reference, we identify important features in broad food and agricultural production and food systems: dairy, food animals, row crops, fruits and vegetables, and environmental resources (water). This analysis explores the relevant concepts of cyber biosecurity from food production to the end product user (such as the consumer) and considers the integration of diverse transportation, supplier, and retailer networks. We describe common challenges and unique barriers across these systems and recommend solutions to advance the role of cyber biosecurity in the food and agricultural sectors

Transglutaminase activation in neurodegenerative diseases

The following review examines the role of calcium in promoting the in vitro and in vivo activation of transglutaminases in neurodegenerative disorders. Diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease exhibit increased transglutaminase activity and rises in intracellular calcium concentrations, which may be related. The aberrant activation of transglutaminase by calcium is thought to give rise to a variety of pathological moieties in these diseases, and the inhibition has been shown to have therapeutic benefit in animal and cellular models of neurodegeneration. Given the potential clinical relevance of transglutaminase inhibitors, we have also reviewed the recent development of such compounds

Faculty Development- Is Some Better Than None?

Introduction: With the advent of competency-based medical education there is an emphasis on formative workplace based assessment. The quality of these assessments is a concern for medical educators and their trainees. Faculty development (FD) strategies to improve assessment quality have resulted in some success. However, few faculty participate, and those who do are likely more motivated to improve, making it difficult to demonstrate a conclusive benefit. To address these weaknesses, we designed a FD initiative to improve the quality of completed in-training evaluation reports (ITERs). All faculty within a division participated. We hypothesized that clinical supervisors would improve their ITER quality based on feedback, regardless of their own motivation to do so, with a simple, point-in-time intervention. Methods: In this three-phase study, two independent raters used the Completed Clinical Evaluation Report Rating (CCERR) to assess the quality of ITERs completed by all faculty in the Division of Orthopedic Surgery at the University of Ottawa. In phase one, ITERs from the previous nine months were evaluated. In phase two, the participants were aware that their ITERs were being evaluated, but they did not receive feedback. In phase three, participants received regular feedback on their performance in the form of their mean CCERR scores. Mean CCERR scores from the different phases of the study were compared. Results: CCERR scores were similar for all three phases (one: 17.56 ± 1.02, two: 17.65 ± 0.96, three: 17.54 ± 0.75, p=0.98). Discussion and Conclusions: There was no evidence in our study that participants' improved their ITER quality despite being aware that they were being evaluated and/or receiving feedback. Potentially, this was related to a lack of motivation. Alternatively, the intensity and/or frequency of the feedback may have been inadequate to create change. These results raise concerns that some faculty development may not necessarily be better than none