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GLC-ASPET Speaker Abstracts 2011

PKC regulation of cys-loop receptors in alcohol and nicotine addiction
Robert Messing
Gallo Center, University of California at San Francisco

Alcohol and nicotine addiction are major public health problems with an estimated combined annual cost of over $400 billion dollars in the US alone. Given the magnitude of the problem, it is surprising that there are few pharmacotherapic agents available to treat patients with these disorders. My laboratory is focused on identifying biochemical mechanisms involved in drug action that control self-administration of these drugs in animals. This lecture will focus on studies of GABAA and nicotinic cholinergic receptors, members of the cysloop superfamily of receptor-operated channels, and their regulation by members of the protein kinase C (PKC) family of serine-threonine kinases. I will discuss our work with PKC epsilon and PKC delta in the regulation of GABAA receptor trafficking and sensitivity to alcohol, and recent studies on PKC epsilon regulation of alpha 6-containing nicotinic receptors in responses to nicotine. These studies highlight PKC epsilon as a potential drug target for the treatment of co-morbid alcohol and nicotine addiction.

Why is PDE10A inhibition predicted to be antipsychotic?
Christopher J Schmidt, Ph.D.
Neuroscience Research Unit
Groton, CT 06340

The psychosis associated with schizophrenia is the result of excessive dopaminergic activity within the striatum, the primary nucleus of a subcortical circuit involved in the regulation of cortically initiated responses to environmental cues. It is at the level of the striatum that cortically selected actions are inhibited or reinforced by ascending dopaminergic inputs from the midbrain. Environmental cues predictive of positive or rewarding outcomes are known to increase striatal dopamine release thereby enhancing the activity of those corticostriatal connections selecting behaviors in pursuit of that outcome while simultaneously inhibiting conflicting behaviors. Cues predicting negative outcome or the loss of a reward result in reduced dopaminergic signaling and favor the selection of avoidance behaviors. The elevated striatal dopamine release 
found in schizophrenia explains why inappropriate behaviors are not inhibited but rather reinforced and maintained despite their potentially negative consequences. Antipsychotic agents block the action of 
dopamine at D2 receptors to allow cortical drive of the striatal neurons involved in the suppression of unwanted behaviors. PDE10A is a dual substrate phosphodiesterase expressed at very high levels in the 
medium spiny neurons (MSNs) of the striatum where it regulates the interaction of glutamatergic and dopaminergic signals by controlling intracellular levels of cAMP and cGMP. Studies conducted in our laboratories confirm that the inhibition of PDE10A can directly oppose the actions of dopamine at D2 receptor on MSNs to enhance their response to cortical drive. We demonstrate that PDE10A inhibition reproduces a majority of the therapeutically relevant biochemical effects of D2 receptor antagonists and is therefore predicted to exhibit antipsychotic activity in the clinic.

MAP kinase phosphatases as therapeutic targets for human diseases
Anton M. Bennett
Yale University School of Medicine, Department of Pharmacology and Program in Integrative Cell Signaling and
Neurobiology of Metabolism, 333 Cedar Street, New Haven, CT 06520

The mitogen-activated protein kinases (MAPKs) have been implicated in a number of pathologies including cancer, obesity, type 2 diabetes, cardiovascular disease, inflammatory and neurological diseases. These observations suggest that the MAPKs, and those signaling components that regulate them, can serve potentially as therapeutic targets to combat these complex diseases. The MAPK phosphatases (MKPs) mediate the dephosphorylation and inactivation of the MAPKs in response to growth factors, stress and electrophysiological stimuli. Therefore, the actions of the MKPs play an important role in the maintenance of physiological MAPK-mediated signaling. Although much work on the MKPs in recent years has demonstrated that this family of enzymes plays essential roles in regulating MAPK-dependent processes a role for the MKPs in disease progression is less clear. We have been investigating the actions of the MKPs in metabolic, inflammatory and musculoskeletal diseases. This work has suggested that the MKPs may play an important role in the progression of obesity and type 2 diabetes, in addition to musculoskeletal disorders such as the muscular dystrophies. We will highlight recent work from this laboratory to support the notion that the MKPs potentially represent meaningful therapeutic targets for the treatment of certain human diseases.

Akt’s functions in cancer and diabetes: Implications for therapy
Nissim Hay
Department of Biochemistry and Molecular Genetics
The University of Illinois at Chicago

The serine/threonine kinase Akt, also known as protein kinase B, is an evolutionarily conserved downstream effector of Insulin/IGF1 receptor and PI3K. In recent years Akt attracted a substantial attention largely because of its frequent hyperactivation in human cancers. However, the most conserved function of Akt is in the regulation of metabolism, and glucose homeostasis at cellular and organismal levels. Using genetics in mice we delineated the in vivo functions of the three Akt isoforms. We showed that Akt is required for oncogenic transformation, and that the deficiency of Akt1 in mice is sufficient to render them resistant to cancer. The function of Akt in cell proliferation and oncogenic transformation is coupled to its role in cellular energy metabolism. Finally, we delineated the functions of Akt isoforms in glucose homeostasis and in diabetes. We analyzed the contributions of the 3 Akt isoforms to diabetes. The diabetic phenotype in Akt-deficient mice is associated with reduced circulating leptin levels, and restoring leptin levels restores normal glucose homeostasis in diabetic Akt-deficient mice.

GLC-ASPET Young Investigator Symposium 2011

Adaptive variation regulates the expression of the human SGK1 gene in response to glucocorticoids
Francesca Luca, PhD
Department of Human Genetics, University of Chicago, Chicago, IL 60637

Glucocorticoids (GCs) are steroid hormones that mediate the response to stress. GCs modulate a large number of biological processes (e.g.: inflammation) and are widely used as pharmacological agents in the treatment of inflammatory diseases, and cancer (e.g. leukemia). GC action is largely mediated by the interaction with the GC receptor (GR), which activates a transcriptional cascade by regulating gene expression of direct targets. The Serum and Glucocorticoid-regulated Kinase1 (SGK1) gene is a target of the GR and is central to the stress response in many human tissues. Because environmental stress varies across habitats, we hypothesized that natural selection shaped the geographic distribution of genetic variants regulating the level of SGK1 expression following GR activation. By combining population genetics and molecular biology methods, we identified a variant with marked allele frequency differences between populations of African and European ancestry and with a strong correlation between allele frequency and latitude in worldwide population samples. This SNP is located in a GR-binding region upstream of SGK1 and within a predicted binding site for Oct1, a transcription factor known to interact with the GR. Using ChIP assays, we showed that both GR and Oct1 bind to this region and that the ancestral allele binds the GR-Oct1 complex more efficiently than the derived allele. Finally, using a reporter gene assay, we demonstrated that the ancestral allele is associated with increased GC-dependent gene expression relative to the derived allele. Our results suggest a novel paradigm in which hormonal responsiveness is modulated by sequence variation in the regulatory regions of nuclear receptor target genes. Identifying such functional variants may shed light on the mechanisms underlying inter-individual variation in response to environmental stressors and to hormonal therapy, as well as in the susceptibility to hormone dependent diseases.

SSRI antidepressants potentiate psychostimulant (Ritalin)-induced gene regulation: risk for enhanced
addiction liability?
Vincent Van Waes, PhD
Department of Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science /
The Chicago Medical School, North Chicago, IL 60064
Current Address: Laboratoire de Neurosciences Integratives et Cliniques, Université de Franche-Compté –
Besancon, France ; Email:

The use of psychostimulants such as methylphenidate (Ritalin), both in the treatment of attention-deficit hyperactivity disorder and as “cognitive enhancers” in the healthy, has increased considerably over the past decades. Methylphenidate, like cocaine, acts by blocking the reuptake of dopamine and norepinephrine. However, unlike cocaine, methylphenidate does not affect serotonin. Studies show that serotonin contributes to cell-signaling effects of cocaine. This may explain why methylphenidate mimics some but not all of the molecular effects of cocaine. We investigated whether serotonin-enhancing medications such as selective serotonin reuptake inhibitors (SSRIs; fluoxetine, citalopram) modify molecular and behavioral effects of methylphenidate. We assessed molecules known to be upregulated by cocaine and other addictive drugs: the transcription factors zif 268 and c-fos, and the neuropetides substance P and dynorphin in the striatum. Our results show that fluoxetine and citalopram robustly potentiate methylphenidate-induced gene regulation throughout the striatum. This potentiation was most pronounced in the sensorimotor striatum. Significant but smaller effects were also seen in subregions of the nucleus accumbens. At the behavioral level, SSRIs did not affect methylphenidate-induced locomotion, but potentiated stereotypies. Further studies showed that methylphenidate+fluoxetine treatment triggers robust reinstatement of cocaine seeking behavior in an animal model of relapse. Together, these findings show that the molecular effects of the methylphenidate+SSRI combination mimic cocaine effects more closely than those of methylphenidate alone, and suggest that SSRI antidepressants potentiate the addiction liability of methylphenidate. This is of concern as methylphenidate+SSRI concomitant therapies are indicated in several neuropsychiatric disorders, and coexposure may also occur with cognitive enhancer use by patients on SSRIs.

Integrin Crosstalk in Endothelial Cells Is Regulated by Protein Kinase A and Protein Phosphatase 1
Annette M Gonzalez, PhD
Department of Pathology, Northwestern University, Chicago, IL 60611

My research interest is in examining integrin-laminin interactions in endothelial cells and how this impacts adhesion and blood vessel development in tumors. Angiogenesis is a complex process that not only depends on growth factors and their receptors but is also influenced by integrin-extracellular matrix interactions. We have identified the alpha 4 laminin, a component of the basement membrane of blood vessels as a ligand for αvβ3 integrin in endothelial cells. We have mapped the binding site of αvβ3 integrin to the carboxy-terminal, globular domain of the alpha 4 laminin subunit and have shown that the alpha 4 laminin subunit plays a key role in angiogenesis in vivo. Moreover, we have uncovered a novel mechanism by which β1 integrin can negatively cross-talk with αvβ3 integrin and regulate its function. We have shown that crosstalk between β1 integrin and αvβ3 integrin involves protein kinase A and components of the inhibitor-1 pathway. Indeed, a phosphatase and protein kinase A appear to act antagonistically in endothelial cells to modulate αvβ3 integrinmediated cell adhesion to alpha 4 laminin, a process that is likely to be an important regulator of vasculogenesis, angiogenesis and blood vessel homeostasis. We have extended these studies in order to gain a better understanding of how integrins function together to regulate cellular processes including adhesion, migration and proliferation. Our results implicate β1 integrin as a master regulator of endothelial cell behavior and indicate that it exerts its functions, in part, by modulating the activity of protein kinase A.

GLC-ASPET Abstracts 2011: Faculty

Involvement of α2-adrenoceptors and imidazoline receptors in agmatine and BMS182874 induced changes in opioid
analgesia and hypothermia
Shaifali Bhalla, Shridhar V. Andurkar, Anil Gulati
Pharmaceutical Sciences, Chicago College of Pharmacy, Midwestern University, Downers Grove, IL 60515

Background and Objectives: Endothelin-A (ETA) receptor antagonist, BMS182874, potentiates opioid analgesia and prevents analgesic tolerance without affecting body temperature. Clonidine (imidazoline/α2-adrenoceptor agonist) enhances opioid analgesia via α2-adrenoceptors and possibly imidazoline receptors without affecting opioid hyperthermia in rats. An endogenous clonidine-like substance, agmatine also enhances morphine analgesia and blocks morphine hyperthermia in rats. However, analgesic interaction of agmatine and oxycodone, and agmatine’s effect on opioid hypothermia in mice are unknown. Objectives were to determine: 1) effect of agmatine on morphine and oxycodone analgesia and hypothermia with or without BMS182874; 2) involvement of ETA-receptors, α2- adrenoceptors, imidazoline receptors in the effect of agmatine and BMS182874 on opioid actions. Methods: Analgesia was determined by tail-flick latency method of D'Amour and Smith in male Swiss Webster mice. Body temperature was determined using Cole-Palmer colonic thermometer. Parameters were measured for 360min and expressed as Mean±S.E.M. N=6 per group. Results: Both agmatine and BMS182874, significantly enhance morphine and oxycodone analgesia. Combination of agmatine and BMS182874 did not further affect morphine (P=0.2536) or oxycodone (P=0.0883). Agmatine-induced increase in morphine and oxycodone analgesia was blocked by imidazoline/α2- adrenoceptor antagonist idazoxan (P=0.0188; P
increase of oxycodone (P=0.0016) but not morphine analgesia (P=0.4493). α2-adrenoceptor antagonist yohimbine blocked agmatine-induced increase of morphine (P=0.0007) and oxycodone analgesia (P

Ion Channel Phosphorylopathy: a link between genomic variations and human disease
Saverio Gentile
Department of Molecular Pharmacology & Experimental Therapeutics, Loyola University Stritch School of Medicine

Voltage gated ion channel (VGIC) regulate several important physiological events including muscle contraction, brain function and secretion. Mutations that inhibit or up-regulate VGCI activities can dramatically interfere with normal organ function and can often lead to unpredictable organ failure, and therefore poor quality of life, or even death. Many genomic variations that change amino acids in cytoplasmic domains of ion channels have been found associated with several diseases however, no mechanism have been proposed to link these mutations to ion channel malfunction. Phosphorylation exerts significant effects on the biophysical mechanisms of ion channels ranging from changes in current kinetics to changes in ion channel trafficking. We have observed that numerous mutations on ion channels associated with diseases can create or disrupt consensus sites for kinases. We call these events “PHOSPHORYLOPATHIES”. We show here that: 1) A mutation on the Cav1.2 associated with Timothy syndrome creates a consensus site for CAMKII and leads to a change in ion channel behavior; 2) Two mutations of the voltage gated potassium channel Kv11.1 associated with the cardiac arrhythmia Long-QT syndrome create or disrupt consensus sites for specific kinases, and therefore affect ion channel behavior. Understanding Phosphorylopathies is crucial in the process of designing an effective pharmacological strategy.

Membrane bound G Protein Estrogen Receptor (GPER) regulates hERG channel activity in ER-neg breast cancer cells
via PKCalpha. Is Cancer a Channelopathy?
Alexander J Donovan, Roma Osauskas, Saverio Gentile
Department of Molecular Pharmacology & Experimental Therapeutics, Loyola University Stritch School of Medicine

Breast cancer is a major cause of death among women worldwide, and novel strategies need to be developed to lessen its morbidity. Although the mechanism of breast cancer proliferation is poorly understood, it has been established that in cancer cells displaying an estrogen receptor positive (ER-pos) or estrogen receptor negative (ER-neg) phenotype, proliferation may be mediated by an estrogen-dependent mechanism. Current treatments that target the ER have recently proven very promising; nonetheless, ER-neg cancers do not respond to ER-targeted therapies, as they lack canonical a-and-b-ER. Moreover, a significant number of ER-neg tumors manifest a more aggressive course of disease with high rates of metastasis compared to other histological types, making the determination of novel targets associated with ER-neg breast cancer paramount. Interestingly, recent studies have demonstrated that estrogen effects on proliferation of ER-neg breast cancer cells can be mediated by the novel membrane bound estrogen receptor, GPER. However, very little is known about the biochemical signaling cascade activated by estrogen via GPER. The human ether-a-go-go related gene (hERG1) encodes the voltage-gated potassium channel Kv11.1, which is primarily expressed in electrically excitable cells. It is well established that Kv11.1 plays a major role in regulating membrane potential in several tissues including the heart and pancreas. Interestingly, recent research in cancer biology has revealed that Kv11.1 is robustly expressed in cancers of varying histogeneses, including brain, prostate, and breast cancer, but not in adjacent non-neoplastic tissue. It has also been shown that blocking Kv11.1 channel activity inhibits proliferation of human cancer cells, suggesting that this channel function plays a significant role in the biology of cancerous tissue; however, nothing is known about the molecular mechanism regulating Kv11.1 ion channel activity in cancer cells and its contribution in proliferation.

A New & Simplified cAMP Assay to Monitor GPCR-Mediated Changes in Intracellular cAMP
Kevin Hsiao*, Mary Sobol and Said A Goueli
Promega Corp. Madison, WI 53711

G Protein Coupled Receptors (GPCRs) represent a large validated target for drug discovery research. Gs is coupled to activation of adenylate cyclase and Gi is coupled to inhibition of adenylate cyclase while Gq is coupled to activation of phospholipase Cb. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a simplified and quick assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCRs that are linked to activation or inhibition of adenylate cyclase (Gs or Gi). The assay is homogenous, easy (one step before detection reagent add in), fast (30min), HTS formatted with high Z’ (>0.7 at 40nM cAMP), and adaptable to multi plate formats. We have successfully generated EC50 values for agonists and IC50 values for antagonists of Gs coupled receptors that are similar to those reported in the literature. The assay does not require antibodies (which use 2 to 5 hour protocols) or expensive instrumentation (such as HT-FRET readers) for signal detection, and the signal output is relatively stable for a few hours. The detection cAMP range is linear up to 100nM, and the S/B is > 50 folds (384well format, unlike HT-FRET only 3 fold maximum).

The 2011 marine pharmaceuticals pipeline
Alejandro MS Mayer1 and Keith B Glaser2,1
1Midwestern University, CCOM, Downers Grove, IL 60515; 2Abbott Laboratories, Abbott Park, IL 60064

As the renaissance in the pharmacology of marine natural products continues (Glaser and Mayer, Biochemical Pharmacology 78:440-448, 2009), the purpose of this project was to assess the status of the clinical marine pharmaceuticals pipeline in 2011. Leading investigators in marine natural products pharmacology research in academic and pharmaceutical communities were surveyed during 2011. Results were the following: there were three FDAapproved marine-derived drugs in the US market, namely cytarabine (Cytosar-U®, Depocyt®), eribulin mesylate (Halaven®), ziconotide (Prialt®), while vidarabine (Vira-A®) was no longer available and, Trabectedin (Yondelis®) being EU-registered. The clinical marine pharmaceutical pipeline (Mayer et al. TIPS 31:255-265, 2010), consisted of thirteen marine-derived compounds but with three new additions, monoclonal antibodies conjugated to synthetic dolastatin derivatives, that were in Phase I, Phase II or Phase III clinical trials. Finally, the preclinical marine pharmacology pipeline remained a global enterprise with researchers from several countries reporting novel mechanisms of action for multiple marine chemicals (Mayer et al. Comparative Biochemistry and Physiology C, 121(1): 63-72, 2011). We conclude that marine pharmacology research as well as the clinical pharmaceuticals pipeline remains very active in 2011.
Supported by ORSP, Midwestern University

Screening, mode of action studies and profiling of Kinase inhibitors with a universal luminescent ADP detection
Zegzouti Hicham, Alves Juliano, Worzella Tracy, Vidugiris Gediminas, Vidugiriene Jolanta and Goueli Said
Promega Corporation, Madison, WI 53711

Many cellular processes are orchestrated by kinases and the disruption of the intracellular signaling networks governed by these enzymes leads to many diseases like cancer. Because of the importance of studying kinases and their increasing recognition as validated drug targets, there have been intense research interests in the development of technologies that monitor the activity of these enzymes. Until now, radioactivity based methods are the most widely used approaches. Although several other technologies were developed in the last few years, most suffer from a variety of limitations that makes it difficult to address all the needs of kinase basic research, drug screening and profiling with one platform. Here, we describe the use of luminescence to measure kinase activity by quantifying the amount of ADP produced in the reaction. We can achieve the sensitivity and robustness required for measuring activity of different kinase families covering the human kinome. As this technology (ADP-Glo) is universal, it is applicable to all kinds of kinase substrates (peptides, proteins, alcohols, lipids, and sugars). It is as sensitive as a radiometric method, and by using an improved Ultra-pure ATP this assay detects ADP at early stages of enzyme reactions with very high signal to background (SB) ratio. Therefore, the ADP-Glo assay allows significant savings of enzyme usage in kinase assays. The assay is fast, simple, homogenous, does not require washes, antibodies, nor custom synthesized substrates, and because of its versatility, this assay can use kinases that are tagged, native, pure or affinity bound to beads. As the ADPGlo™ technology was used and validated with hundreds of kinases; the assay is now optimized for use with a large panel of complete Kinase Enzyme Systems (KES) that span different families of the human kinome. We also show the results of an optimized protocol for profiling different Kinase inhibitors using ADP-Glo™ and 70 KES. The fact that ADPGlo™ assay offers so many positive attributes makes it an ideal assay for all kinase studies, including basic research, primary and secondary screening, and also profiling of lead compounds.

GLC-ASPET Abstracts 2011: Postdoctoral Fellows

Differential modulation of corticostriatal transmission by phosphodiesterase 9A and 10A activation
1Department of Neuroscience, Rosalind Franklin University of Medicine and Science / The Chicago Medical School, North
Chicago, IL 60064; 2Pfizer, Inc.

The cyclic nucleotides cAMP and cGMP are critically involved in the modulation of striatal synaptic transmission. Striatal medium-sized spiny neurons (MSNs) contain high levels of cyclic nucleotide phosphodiesterases (PDEs) involved in the metabolism of cAMP and cGMP. Inhibition of the dual substrate enzyme PDE10A increases the responsiveness of MSNs to cortical input. This effect appears to be more robust in striatopallidal MSNs and depends largely on cGMP signaling. While the cGMP specific enzyme PDE9A appears to regulate tonic levels of cGMP, the impact of this regulation on corticostriatal transmission is not known. Therefore, the current study compared the effects of inhibiting PDE10A and PDE9A on corticostriatal transmission in urethane anesthetized rats. Spike activity recorded in the dorsocentral striatum was evoked by electrical stimulation of the ipsilateral frontal cortex in rats treated with either vehicle or well characterized doses of the selective PDE10A and PDE9A inhibitors TP-10 (3.2 mg/kg, s.c.) and PF-4181366-42 (10.0 mg/kg, s.c.), respectively. To enable comparisons of cortically-evoked activity across groups, stimulation currents were titrated to an intensity which reliably evoked spike activity in ~50 % of trials, and potential drug-induced changes in this measure were determined. Using this approach, we found that MSNs recorded in animals treated with TP-10 required lower stimulation current intensities to reach 50% spike probability. The onset latency of evoked spikes was also significantly decreased in TP-10 treated rats as compared to vehicle-treated controls. These findings are consistent with our previous studies showing that inhibition of PDE10A activity increases the responsiveness of MSNs to corticostriatal input. Interestingly, no significant changes in cortically-evoked activity were observed following treatment with PF-4181366-42. These results suggest that PDE10A and PDE9A may regulate cGMP signaling in functionally distinct subcellular compartments within MSNs. While the function of PDE9A remains to be clarified, our data indicate that the pool of cGMP associated with this PDE9A regulation does not play an obvious role in modulating corticostriatal transmission. These findings have implications for the consideration of PDE9A and PDE10A as novel drug targets for treating neurological and psychiatric disorders.

Mixed Lineage Kinase-3 phosphorylates α-Synuclein, leads to its aggregation: Possible Mechanism of α-Synuclein
toxicity in Parkinson’s disease.
Suneet Mehrotra1, Gautam Sondarva1, Velusamy Rangasamy1, Rajakishore Mishra1, David Freeman2, Edward Campbell2
and Ajay Rana1
1Department of Molecular pharmacology and Therapeutics, Stritch School of Medicine, Loyola University Chicago,
Maywood, Illinois 60153; 2Department Microbiology and Immunology, Stritch School of Medicine, Loyola University
Chicago, Maywood, Illinois 60153

Mixed Lineage Kinase3 (MLK3) is a MAP3K member. MLKs are unique in that all family members contain signature sequences of both Ser/Thr and Tyr kinases within their catalytic domains and thus they are called mixed lineage kinases. The role of MLK in neuronal cell death pathways was unknown, until recently when a specific inhibitor of MLK family, CEP-1347 was shown to prevent dopaminergic neuronal cell death in a MPTP model of Parkinson's disease (PD). Alpha-Synuclein (α-syn) is a protein whose function in the healthy brain is not currently known. The role of α-syn in PD is of great interest as it is a major constituent of Lewy bodies (LB), protein clumps that are the pathological hallmark of PD. Phosphorylation of α-syn leads to its aggregation and cause LB and related synucleinopathies. The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. We for the first time report that MLK3 phosphorylates α-syn at Thr 22 and Thr 72 sites. The mutants of this phosphorylation inhibit α-syn aggregation. The pan MLKs inhibitor CEP-1347 inhibited α-syn aggregation upon MPP+ treatment in N27 cells, over expressing human α-syn. Thus, here we submit evidence that MLK3 causes phosphorylation of α-syn and promotes its aggregation, which might leads to α-syn-induced toxicity in PD.

SSRIs potentiate methylphenidate-induced gene regulation in the striatum: transcription factors vs. neuropeptides
Vincent Van Waes, Betsy Carr, Joel A Beverley and Heinz Steiner
Department of Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science/The Chicago
Medical School, North Chicago, IL 60064

The psychostimulant methylphenidate (Ritalin) is used in the treatment of attention-deficit hyperactivity disorder and as a “cognitive enhancer” in the healthy. Methylphenidate, like cocaine, acts by blocking the reuptake of dopamine and norepinephrine. However, unlike cocaine, methylphenidate does not affect serotonin. It is known that serotonin contributes to cell-signaling and behavioral effects of cocaine. Therefore, lack of a serotonin effect may explain why methylphenidate mimics some but not all of the molecular effects of cocaine. We investigated whether enhancing serotonin action by adding a selective serotonin reuptake inhibitor (SSRI) antidepressant, fluoxetine (Prozac), to methylphenidate modifies the molecular effects of methylphenidate. Periadolescent rats were treated with fluoxetine (5 mg/kg, i.p.) in conjunction with methylphenidate (2-5 mg/kg), and gene expression in the striatum was measured by in situ hybridization histochemistry. We assessed molecules known to be upregulated by cocaine and other addictive drugs: the transcription factors zif 268 and c-fos, and the neuropetides substance P and dynorphin. Our results demonstrate that fluoxetine robustly potentiates methylphenidate-induced expression of zif 268 and c-fos throughout the striatum. This effect was most pronounced in the sensorimotor striatum. Fluoxetine also potentiated methylphenidate-induced substance P and dynorphin expression. While the potentiation of neuropeptide expression was more moderate than that of transcription factors, both occurred in the same striatal regions. These results show that the molecular effects of the methylphenidate plus SSRI combination mimic cocaine effects more closely than those of methylphenidate alone. Methylphenidate plus SSRI concomitant therapies are indicated in several neuropsychiatric disorders, and co-exposure may also occur with cognitive enhancer use by patients on SSRIs. The here observed molecular changes are considered to reflect addiction-related neuroadaptations. Our findings thus suggest that SSRI antidepressants may potentiate the addiction liability of psychostimulants such as methylphenidate.

Serotonin 5-HT1A receptor activation inhibits HIF-1α expression in a rat embryonic serotonergic cell line
Nichole L Dudek, Jaime Vantrease and Karie E Scrogin
Department of Pharmacology, Loyola University Chicago School of Medicine, Maywood, IL

Slowed maturation of serotonergic cells of the caudal raphe has been implicated in sudden infant death syndrome. Little is known about how the development of these cells is regulated. Hypoxia-inducible factor 1 (HI-F1 is a short lived transcription factor that is stabilized by hypoxia and certain ligand-receptor interactions, and promotes cell survival. Here, we determined if the serotonin 5-HT1A receptor (5-HT1AR) agonist, 8-OH-DPAT, affects HIF-1 levels in RN46A cells, a neuronal cell line derived from rat embryonic raphe neurons that endogenously express 5-HT1ARs. Differentiation of RN46A cells at 37ºC results in detectable HIF-1α levels and increased expression of tryptophan hydroxylase (TPH), the rate limiting enzyme in serotonin synthesis. Treatment of differentiated RN46A cells with 8-OHDPAT (1μM) transiently decreased HIF-1α levels (-60% at 10 min, P

GLC-ASPET Abstracts 2011: Graduate Students

Short-term exposure of leukemia cells to chemotherapeutic agents is associated with changes in microRNA
Tsui-Ting Ho, Ahmet D. Arslan, Xiaolong He, William T. Beck
Department of Biopharmaceutical Sciences, College of Pharmacy, and Cancer Center, University of Illinois at Chicago,
Chicago, IL, 60612

The acquisition of resistance to anticancer drugs is a key obstacle to successful cancer therapy. An increasing number of studies have investigated the roles of microRNAs (miRs) in drug resistance. We reported that the upregulation of miR-135b/-196b correlated positively with acquired drug resistance as well as expression of the ATP-binding cassette (ABC) transporter ABCB1 in the human T-cell leukemic CCRF-CEM cell line. We found similar increases in miR-135b/-196b in other cell lines derived from leukemias, but not from solid tumors, suggesting that induction of expression of these miRs may be histiotype-specific. miR-196b maps between the HoxA9 and HoxA10 genes. We observed upregulation of HoxA9 mRNA in relation to short-term chemotherapeutic drug exposure in CCRF-CEM cells, indicating that the expression of miR-196b is linked to HoxA gene transcription. To investigate the kinetics of expression of miR-135b/-196b, ABCB1 and ABCC1, we challenged CCRF-CEM cells repeatedly with etoposide for 48 h with a 3-day drugfree incubation in between. We found that changes in miR-135b/-196b expression were paralleled by expression of the ABCB1 gene, but not the ABCC1 gene. These results suggest that miR-135b and -196b are involved not only in the initial response of cancer cells to chemotherapeutic challenge, but also in the development of chemoresistance. We subsequently measured expressions of miR-135b/-196b, ABCB1 and ABCC1 in CCRF-CEM cells exposed to repeated drug challenge after either 15 or 20 passages in drug-free medium. Treatment of these previously-exposed cells again with etoposide resulted in unexpected attenuation of miR-135b/-196b and ABCB1 expression. We further determined that the etoposide IC50 in these cells had increased from 300 nM to 1 μM. Our results suggest that induction of miR-135b/-196b after drug exposure is transient and offer insights into the establishment of the drug-resistant phenotype which seems to re-set the IC50 to higher levels.

Single Nucleotide Polymorphisms Associated With Pain in Sickle Cell Disease
Ellie Jhun1, Ying He1, Robert E Molokie2,3, A Kyle Mack4,5, Diana J Wilkie6, Zaijie Jim Wang1
1Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago (UIC); 2Jesse Brown Veteran’s
Administration Medical Center; 3Division of Hematology/Oncology, College of Medicine, UIC; 4Department of Pediatrics,
Northwestern University Feinberg School of Medicine; 5Children’s Memorial Center for Cancer and Blood Disorders;
6Department of Biobehavioral Health Science, College of Nursing, UIC. Chicago, IL, 60607

Background: Pain is a major complaint in patients with sickle cell disease (SCD). SCD pain is characterized by episodes of acute pain that is responsible for the majority of ER visits by SCD patients and by persistent chronic pain that is detrimental to quality of life. Pain severity and frequency vary signifiantly in SCD patients. We hypothesize that genetic polymorphisms may account for some of these variations. In the study, we applied the candidate gene approach to examine the role of several single nucleotide polymorphisms (SNPs). Methods: Adult and pediatric sickle cell subjects were recruited during routine outpatient clinic visits. Subjects entered answers to computerized pain questionnaires (PAINReportIt ®), from which composite pain index (CPI) scores were calculated. Blood samples were collected for DNA 
extraction and genotyping according to published methods (PCR-RFLP). Data were analyzed using Pearson’s chi-square and fisher’s exact. The Benjamini-Hochberg method was used to correct for multiplicity. Results: The following SNPs were analyzed in this study: OPRM1 rs1799971, G691C rs2075572; COMT rs4680; DRD3 rs6280; and TRPV-1 rs222747 and rs224534. These SNPs were chosen based on previous genetic or pharmacological studies. Allelic frequencies of TRPV-1 rs222747 were significantly associated with CPI scores. The minor allele was associated with lower CPI scores. The DRD3 major allele was associated with fewer utilizations per year for both genotype and allele. Two other SNPs, rs2075572 and rs4680, were approaching significance when stratified into adults and pediatrics; however, they were under powered. Conclusions: Our candidate genetics approach is preliminary, yet powerful in identifying potential genetic influence on pain variations either in severity (CPI) or frequency (utilization). Two SNPs (rs6280 and rs222747) are significantly associated with pain phenotypes in patients with SCD.

Utility of Bromoindirubin -3 Oxime (BIO) in the Generation of Progenitor Cells for Regenerative and Transplantation
Erin E. Kohler, Asrar B Malik and Kishore K Wary
Department of Pharmacology, University of Illinois at Chicago, Chicago, IL-60612

RATIONALE: We have previously shown that Wnt signaling induces increased expression of NANOG, a transcription factor involved in the maintenance of ESC pluripotency and self-renewal, to control proliferation and neovascularization by up-regulating VEGFR-2 (also referred to as Flk1) and Cyclin-D1 in ECs (1). Given that the Wnt and the transforming growth factor-1 (TGF-1α) signaling pathways induce dedifferentiation of endothelial cells (ECs) into mesenchymal-like cells, increased NANOG expression may serve as a phenotypic switch in these cells. GOAL: The goal of this study is to address the role of BIO, an inhibitor of GSK-3β to transiently and reversibly induce increased expression of NANOG in a population of normal ECs to generate large numbers of endothelial progenitor cells (EPCs) for regenerative and transplantation medicine. RESULTS: In ECs, BIO induced increased expression of NANOG and several mesenchymal and hemangioblastic cell markers, while it decreased expression of endothelial cell markers von Willebrand Factor (vWF) and PECAM-1 (CD31). Increased NANOG expression induced characteristics of mesenchymal/stem-like cellular behavior in hanging drop assays. Importantly, BIO stimulation increased proliferation and migration of ECs in vitro and angiogenesis in vivo. Furthermore, microscopic analyses indicated the ability of BIO to induce asymmetric cell division in these cells, a hallmark of stem cells. While NANOG knockdown inhibited the ability to divide asymmetrically and to form cellular aggregates in hanging drop assays, and angiogenesis in these BIO treated cells. CONCLUSION: These results, for the first time, indicate the capacity of BIO to induce dedifferentiation of ECs into endothelial progenitor/stem-like cells by increasing the expression of NANOG and NANOG target genes. Thus, we propose that BIO could become useful not only for inducing neovascularization of ischemic tissues, but also for generating a large number of cells for transplantation and regenerative medicine.

The effects of methamphetamine treatment on the blood brain barrier and cerebral vasculature
SM Kousik, SM Graves, TC Napier and PM Carvey
Department of Pharmacology and Center for Compulsive Behavior and Addiction, Rush University, Chicago, IL 60612

Methamphetamine (meth) is a potent psychostimulant shown to induce neurotoxicity. The exact mechanism(s) of meth-induced neurotoxicity is unknown. We sought to evaluate meth-induced blood-brain barrier (BBB) alterations as well as possible cerebral vasculature changes. To do so, rats were treated ip with 0.9% saline, 3 or 9 mg/kg meth, or trained to self-administer meth chronically. All animals were anesthetized, and transcardially perfused with fluorescein isothiocyanate labeled albumin (FITC-LA), a marker of vascular integrity. Rats given acute 3 or 9 mg/kg meth had higher FITC-LA optical density measurements in the prefrontal cortex and nucleus accumbens shell as compared to other brain regions, and to these regions in saline treated rats. This outcome suggested that BBB dysfunction was region selective. Compared to controls, the dorsal striatum (dStr) of rats from all three meth treatment groups uniquely exhibited a striking absence of FITC-LA (verified with FITC-LA optical density measurements) suggesting that meth induces region-specific vasoconstriction. Selective cerebral hypoxia was further quantified immunohistochemically using hypoxia inducible factor 1α (HIF1α). These compelling findings concur with clinical reports of reduced regional cerebral blood flow (rCBF) in the sensorimotor regions of the striatum, while the associative and limbic regions maintain normal rCBF. Selective sensorimotor regional reduced rCBF may destroy the dopamine terminals in the striatal region most affected by Parkinson’s disease (PD). Thus, we measured tyrosine hydroxylase (TH) optical density in the striatum and cell counts in the substantia nigra to determine if any retrograde neuronal loss occurred with meth treatment. We observed a reduction in TH+ terminals in the striatum, which is in keeping with recent clinical studies suggest that meth-induced neurotoxicity may increase vulnerability to develop PD. 
Our study represents the first pre-clinical model of meth-induced cerebral vascular changes in the dStr as a new mechanism of meth-induced neurotoxicity that may provide a novel model for meth-induced PD risk and offer insight into how dopamine neuron loss may occur in idiopathic PD. Supported by the Center for Compulsive Behavior and Addiction, Rush University; all work was performed at Rush University.

Are p90 ribosomal S6 kinases involved in the mechanism of thromboxane B2 inhibition by the marine β-carboline
Manzamine A?
Joseph Lach, Mary L Hall and Alejandro MS Mayer
Midwestern University, CCOM, Downers Grove, Illinois 60515

Manzamine A (MZA is a β-carboline alkaloid first reported from the Okinawan marine sponge genus Haliclona in 1986.
We have reported that in vitro Manzamine A potently inhibited LPS-activated rat microglia thromboxane B2 (TXB2) (IC50

A non-GPCR dependent role for P-Rex1 in endothelial cells
Ram P. Naikawadi1, Ni Cheng1, Dianqing Wu2 and Richard D Ye1
1Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612; 2Department of Pharmacology, Yale
University school of Medicine, New Haven, CT 06520

Maintenance of vascular endothelial integrity is of great importance to homeostasis of vital organ functions. The small GTPase Rac is one of the key signaling mediators for vascular endothelial functions, but how Rac activation is regulated under various pathophysiological conditions remains incompletely understood. Using gene deletion and knockdown approaches, we investigated the potential role of the phosphoinositide- and G protein b subunitsregulated guanine nucleotide exchange factor P-Rex1 in TNF-a stimulated endothelial cells. P-Rex1, previously found in neutrophils and neurons, is also expressed in endothelial cells. In cultured human lung microvascular endothelial cells (HLMVECs), TNFa stimulation resulted in a decline in transendothelial electrical resistance (TER). Small interference (si) RNA-mediated knockdown of P-Rex1 markedly reversed the loss of TER and reduced intercellular gap formation. HLMVECs receiving PRex1-specific siRNA also displayed significantly reduced Rac activation, reactive oxygen species (ROS) production and phosphotyrosine dependent VE-cadherin phosphorylation upon stimulation with TNF-a, compared to HLMVECs receiving scrambled siRNA. TNF-a stimulated P-Rex1 membrane translocation and the resulting Rac activation is dependent on PI3K. Removal of P-Rex1 from endothelial cells produced a more significant reduction in neutrophil transendothelial migration when compared to removal of P-Rex1 from neutrophils. Moreover, significantly less neutrophils was found in the bronchoalveolar fluid of P-Rex1 deficient mice compared to WT mice after intratracheal instillation of TNF-a. Mice lacking the P-Rex1 gene are also resistant to TNF--induced lung edema, suggesting that PRex1 is a critical mediator of vascular permeability. These results demonstrate for the first time a pivotal role of endothelial P-Rex1 in the modulation of vascular endothelial integrity, suggesting the possibility for targeting endothelial P-Rex1 in the control of lung edema and neutrophil infiltration to inflammatory tissues.

Inhibition of the NF-κB pathway by the curcumin analogs EF24 and EF31: anti-inflammatory and anti-cancer
Anlys Olivera, Andrew P Brown, Terry W Moore, Aiming Sun, Fang Hu, Dennis C Liotta, James P Snyder, Hyunsuk Shim,
Adam I Marcus, Andrew H Miller, Thaddeus WW Pace
Emory University, Atlanta, Georgia, 30322

The inflammatory transcription factor, nuclear factor kappa B (NF-κB), is a critical link between chronic inflammation and cancer. Therefore, we have endeavored to develop novel anti-inflammatory compounds that target NF-κB. Data indicate that curcumin, an ingredient of the curry spice turmeric, acts as a NF-κB inhibitor. EF31 and EF24 are structurally related to curcumin but exhibit increased potency and bioavailability. To examine the relative activity of these compounds in-vitro, mouse RAW264.7 macrophages were treated with EF31, EF24, curcumin (1–100μM) or vehicle (DMSO 1%) for 1 hour. NF-κB pathway activity was assessed following treatment with lipopolysaccharide (LPS) (1μg). LPS induced NF-κB activity in nuclear extracts peaked at 15 minutes, whereas expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α peaked at 3 hours. Potent inhibition of NF-κB DNA-binding was observed at concentrations of 5-10μM for EF31, 30-50μM for EF24, and 50-100μM for curcumin. In addition, EF31 exhibited significantly greater inhibition of NF-κB nuclear translocation as well as the induction of downstream inflammatory mediators including pro-inflammatory cytokine mRNA and protein. Moreover, EF31 exhibited significantly greater inhibition of IκB kinase β compared to EF24. Finally, EF31 demonstrated potent toxicity in NF-κB-dependent cancer cell lines while having minimal and reversible toxicity in RAW264.7 macrophages. These data indicate that EF31 is a more potent inhibitor of NF-κB activity than either EF24 or curcumin while exhibiting both anti-inflammatory and anticancer activities. Curcumin analogs including EF31 may represent a novel approach to treat cancer by hindering the effects of inflammation on the initiation and progression of tumors. Thus, EF31 represents a promising curcumin analogue for further therapeutic development.

Cleavage of the Pro-Survival Protein Intersectin-1s by Granzyme B Induces Endothelial Cell Proliferation via p38
MAPK and Elk-1 Transcription Factor Activation
Monal Patel, Minhua Wang and Sanda Predescu
Rush University, Department of Pharmacology, Vascular Biology Section, IL 60612

Inflammation associated with various human diseases and the changes in endothelial cells (ECs) phenotype to support various phases of the inflammatory process are key patho-biological players in lung vascular remodeling and subsequent vascular disorders. In inflammatory states, like rheumatoid arthritis, pulmonary hypertension, connective tissue disease, the number of inflammatory cells, including T lymphocytes, is increased. The CD8+ cytotoxic T cells contain perforins and granzyme B (GrB) which are released from the cytotoxic granules to induce target cell death. The cytotoxic protease GrB can initiate ECs apoptosis by cleaving caspases and other proteins important for proliferation and cell survival. Using in vitro and in vivo approaches we now show that intersectin-1s (ITSN-1s), an essential protein for lung ECs survival, is cleaved by GrB at the sequence IDQD271K and generates an N-terminal cleavage product (GrBEHITSN), of 28 kDa. Significantly, expression of myc-GrB-EHITSN in ECs causes marked increase in cell proliferation, as indicated by bromodeoxiuridine (BrdU) incorporation detected with anti BrdU Ab. Additional Western blot analyses and Enzyme-Linked Immunosorbent Assay applied on lysates or nuclear extracts respectively, prepared from control and myc-GrB-EHITSN-transfected cells, indicate that GrB-EHITSN may be implicated in controlling cell proliferation via persistent activation of p38 MAPK and Elk-1 transcription factor. While the C-terminal GrB cleavage fragment has dominant negative effects on Ras/Erk1/2 survival signaling, expression of GrB-EHITSN and downstream p38 activation further inhibits Erk1/2 phosphorylation. Altogether, our studies provide evidence that loss of full-length ITSN-1s, the proliferative properties of GrB-EHITSN, selective activation of p38 MAPK and downstream Elk1, as well as the modulatory effect of ITSN-1s GrB cleavage products on p38/Erk1/2 MAPK signaling may provide an advantage for p38 MAPK signaling cascade favoring the selection of a hyper-proliferative EC phenotype. 
Overexpression of PKCα in breast cancer cells induces migration through p120-catenin transcriptional
Bethany E. Perez White*, Huiping Zhao and Debra A. Tonetti
University of Illinois at Chicago, Chicago, IL 60612

Several factors have been identified that influence epithelial-mesenchymal transition (EMT) in breast cancer cells. We previously reported that overexpression of PKCα in breast cancer predicts tamoxifen resistant secondary tumors (Tonetti, 2003). In breast cancer cells, stable overexpression of PKCα (T47D/PKCα) leads to hormone-independent growth as well as tamoxifen resistance (Chisamore, 2001). In our current study we report that PKCα overexpression increases the metastatic potential of breast cancer cells through induction of a nonclasssical EMT. T47D/PKCα cells are morphologically distinct from T47D/neo cells and do not retain the cuboidal structure of epithelial breast cancer cells. Overexpression of PKCα resulted in significantly enhanced migratory capabilities in the Boyden chamber assay. PKC activation by phorbol 12-myristate 13 acetate further enhanced migration in T47D/PKCα cells. Pharmacological inhibition with the classical PKC inhibitor Gö6976 reduced the migratory capacity of T47D/PKCα cells. Transient siRNAmediated knockdown of PKCα (120 h) significantly reduced both basal and NIH3T3 fibroblast conditioned mediainduced migration in T47D/PKCα cells. Levels of adherens junction proteins E-cadherin, α-E-catenin, β-catenin and p120-catenin were significantly downregulated in T47D/PKCα cells compared to T47D/neo cells as determined by western blot. Levels of p120-catenin protein were restored after 120 h of PKCα knockdown, while levels of E-cadherin protein did not increase until after 144 h of PKCα knockdown in T47D/PKCα cells. E-cadherin transcripts analyzed by SYBR green RT-qPCR were significantly higher in T47D/PKCα cells compared to T47D/neo control cells while there was no change in expression of β-catenin or α-E-catenin transcripts. Only p120-catenin transcript levels were significantly lower. Treatment with proteasomal inhibitor MG132 induces accumulation of E-cadherin but not p120-catenin. Taken together, these data suggest that PKCα may be responsible for a nonclassical EMT in breast cancer cells through upstream signaling that leads to transcriptional inhibition of p120-catenin and subsequent degradation of E-cadherin. Further, PKCα expression may not only be predictive of tamoxifen-resistance but of increased potential for metastasis, two of the biggest clinical hurdles that preclude the effective management of breast cancer. Further studies are required to define PKCα as a potential therapeutic target or biomarker.

Neuroprotective effect of liraglutide in a rat model of cerebral ischemia
Sagar Shah1, Seema Briyal2 and Anil Gulati2
1Biomedical Sciences Program, College of Health Sciences and 2Department of Pharmaceutical Sciences, Chicago College
of Pharmacy, Midwestern University, Downers Grove, IL 60515

Stroke is a common life-threatening neurological disorder that requires rapid workup and treatment. GLP-1 has been found to protect neurons against oxidative insults. Liraglutide is a GLP-1 analogue approved to treat type 2 diabetes mellitus that crosses the blood brain barrier and may potentially treat cerebral ischemia. The aim of this study was to assess the effect of liraglutide on neurological outcome, oxidative stress and infarct volume in rats subjected to cerebral ischemia by middle cerebral artery occlusion (MCAO) in normal as well as in diabetes. Male Sp.Dawley rats were subjected to cerebral ischemia by MCAO. Animals were pretreated with either vehicle or liraglutide (50 μg/kg, s.c.) for 14 days. At 24 h after occlusion, rats were assessed for neurological and motor function. Then, animals were sacrificed for estimation of oxidative stress markers and infarct volume. In the diabetic group, streptozotocin (45 mg/kg, i.p.) was administered 3 days prior to vehicle/liraglutide injection. Non-diabetic and diabetic rats treated with vehicle showed significant (P

Effects of L-DOPA on risk-taking behaviors in rats
Stephanie Tedford1, Sandra Rokosik1,2 and T Celeste Napier1
1Dept. of Pharmacology, and Center for Compulsive Behavior and Addiction, RUSH University Medical Center, 60607;
2Neuroscience Program, Loyola University Medical Center, Maywood, IL 60153

A subset of Parkinson’s disease (PD) patients treated with dopamine (DA) agonists demonstrates impulse control disorders, such as pathological gambling. These side effects are associated with DA D3 receptor (D3R) preferring agonists including pramipexole (PPX) and are rarely seen with monotherapy using L-DOPA, an indirect DA agonist. To better understand the underlying neurobiology of these disorders, we developed a preclinical model of risk-taking using a probability discounting task in laboratory rats. In this task, a subject chooses between a small positive reinforcer that is always delivered and a large reinforcer that is delivered with varying probabilities. Risky behavior is reflected in a preference for the large, uncertain reinforcer. Our lab has implemented this task using intracranial selfstimulation of the medial forebrain bundle (MFB) as the positive reinforcer and determined that PPX increases risktaking in a rat model of PD and sham controls. For the current study, we aimed to assess the propensity of L-DOPA to induce risk-taking. To model PD, rats were bilaterally injected with 6-OHDA or vehicle into the dorsalateral striatum. The rats were also implanted with an electrode into the MFB. A forelimb step task validated motor deficits reflecting successful DAergic lesions of the striatum, and reversal of these deficits established the L-DOPA dose to be tested in the probability discounting task. Dose-response evaluations verified that Emax for improvements in stepping was reached for 3, 6 and 12mg/kg ip L-DOPA. For the second phase of this study, unlesioned rats were trained on the discounting task, and after a stable risk-taking baseline was established, they were treated with 12mg/kg ip L-DOPA 2X a day for 15 days. Risk-taking was assessed 30 min and 4 hr post injection every other day. Data collected thus far reveal that 12mg/kg L-DOPA does not alter risk-taking in control rats. Ongoing studies are determining the effects of LDOPA in PD-like rats. This work was supported by the Michael J. Fox Foundation, the Parkinson’s Disease Foundation Center Grant, Novel Projects Program Rush University and the Rice Foundation.

NMDA receptor blockade during adolescence disrupts the normative development of prefrontal cortical inhibitory
Thomases D, Cass DK, Caballero A, Tseng KY
Department of Cellular & Molecular Pharmacology, Rosalind Franklin University of Medicine and Science, The Chicago
Medical School, North Chicago, IL 60064

Despite the fact that a developmental disruption of cortical inhibitory circuits is thought to contribute to the adolescent onset of cognitive deficits observed in many major psychiatric disorders (e.g., schizophrenia), the neuronal basis and pathophysiological consequences underlying such changes remains unclear. Among the possible players, the N-methyl D-aspartate (NMDA) receptor is thought to play a crucial role in the maturation of parvalbumin (PV)-positive/fastspiking GABA interneurons in the prefrontal cortex (PFC). We hypothesized that if during development, such interneuronal activity does not become enhanced, prefrontal inhibitory control will be altered at maturity. Here we examine how repeated administration of the NMDA antagonist MK-801 during the periadolescent transition (postnatal day -PD- 35 to 40) changes the normal development of PV-positive interneurons in the PFC. We found that the level of PV-positive interneurons is diminished in the adult (PD>65) PFC when animals were treated with MK-801 during adolescence. No apparent changes in PV-immunoreactivity were observed in rats treated during adulthood (PD 75-80). In addition, electrophysiological analyses of cortical network activity indicate that MK-801-induced developmental dysregulation of interneuronal circuits can elicit a sustained, disinhibited PFC state. Such PFC disinhibition is associated with increased responsiveness as well as abnormal synaptic integration of synchronous inputs from the ventral hippocampus. We next examined the behavioral consequences of the decreased prefrontal interneuronal function by using the discrete paired delayed alternation paradigm to measure deficits in prefrontal-dependent working memory and acquired learning. Adolescent-treated rats displayed marked deficits in acquired learning capabilities, relative to controls when tested in adulthood. Together, these results indicate that glutamatergic activation of PV-positive GABAergic interneurons by NMDA receptors is critical for interneuronal development in the PFC. Impairment of such processes in the PFC could contribute to the periadolescent onset of cognitive deficits observed in schizophrenia and related psychiatric disorders. Supported by Rosalind Franklin University (KYT) and National Institute of Health Grant R01-MH086507 (KYT).

Enduring Alterations in the Prefrontal Cortex After a Single In Vivo Exposure of the HIV-1 Protein Tat
1Department of Pharmacology and 2Neuroscience, and 3Center For Compulsive Behavior and Addiction, Rush University,
Chicago, IL; 4Microbiology, George Mason University, Manassas, VA

HIV-1-infected patients, even with highly active antiretroviral therapy, can develop HIV-associated neurocognitive disorders (HAND). While the neuropathogenesis of HAND is unknown, neurotoxicity of HIV proteins, including HIV-1 Tat (transactivator of transcription), is thought to play a critical role. The prefrontal cortex (PFC), a regulator for cognition and motivated-behaviors, is altered in those with HAND. Our recent studies revealed that Tat enhanced responses of pyramidal cells in the medial PFC to excitatory stimuli; this Tat effect was mediated by over-activated L-type Ca2+ channels with abnormally enhanced Ca2+ influx. For the current study, we hypothesized that a short-term in vivo exposure of Tat at pathophysiological levels will (1) increase the expression of the L-channels, (2) induce astrocytosis, but will not (3) cause neuronal death in the PFC. For these studies, adult rats were given a single unilateral intracerebroventricular (icv) injection of recombinant Tat (80μg/20μl), or its vehicle (n=3/each). Fourteen days after the icv injection, mPFC tissue was harvested and immunohistochemistry (IHC) was conducted. Stereological estimates and optical density were used to quantify Tat-induced increases in (1) L-channels, (2) glial fibrillary acidic protein (GFAP) a marker for astrocytosis, and (3) neuronal nuclear protein (NeuN) a marker for cell death. Our results indicated that the number of cells immunoreactive (ir) for the Cav1.2 Ca2+ L-channels and GFAP were significantly increased within the PFC of Tat-exposed rats compared to vehicle-injected controls. There was no change in the number of cells immunoreactive for NeuN. In summary, neither the astrocytosis nor the upregulation of Ca2+ channels was sufficient to cause the neuronal death that is commonly seen in later time points of HIV infection. However, as Tat can induce hyper-excitability of these pyramidal neurons via L-type Ca2+ channels and these channels are abnormally upregulated, channel blockers may be an efficacious therapeutic target to reduce the impact of HIV-1 proteins on the brain. Supported by the Rice Fdn., McManus Fdn and the Ctr Compul Beh & Addiction, RUMC.

Adolescent rats are more vulnerable to cocaine self-administration than adults
Department of Cellular and Molecular Pharmacology, Rosalind Franklin University of Medicine and Science, North
Chicago, IL 60064

Adolescent rats take cocaine more readily than adults, are sensitive to lower doses, and work harder for access to the drug. Our study shows, for the first time, that adolescents are more sensitive to lower doses of cocaine, and they will work harder to obtain it. Our research is the first to offer scientific evidence that when all opportunities to take drugs are equal, biology alone makes adolescents more likely to use cocaine compared to adults. Adolescence is a period of heightened propensity to develop cocaine addiction in humans. We have previously shown that midbrain dopamine neurons are more active in adolescent rats compared with adults. Given that elevated activity of dopamine neurons is associated with elevated propensity to self-administer cocaine in rats, we tested whether the period of adolescence is associated with higher liability to self-administer cocaine relative to adulthood. Adolescent (PND 42) and adult (~PND 88) rats were compared for their sensitivity to, and motivation for, cocaine using self-administration. Different cohorts of rats were tested either (1) for acquisition of saline or very low to moderate doses of cocaine (75, 150, 600 μg/kg), (2) on a within-session dose-response paradigm (18.75-1200 μg/kg), or (3) on a between-session progressive ratio paradigm (600 μg/kg) in which the ratio was increased every other day (1, 3, 6, 12, 24, 48). In the acquisition tests, adolescents showed higher intake of cocaine than adults at low to moderate doses (150 μg/kg and 600 μg/kg), but did not differ in intake of a very low dose of cocaine (75 μg/kg) or saline. Adolescents and adults did not differ in the within session dose-response paradigm. In the between-session progressive ratio test, adolescents worked more to obtain cocaine than adults, suggesting that adolescents have greater motivation to self-administer cocaine than adults. In conclusion, we show that adolescent rats are more likely to acquire self-administration of cocaine than adults at low to moderate doses of cocaine, and show greater motivation to self-administer this drug. These experiments could help explain the greater addiction liability observed in human adolescents relative to adults.

GLC-ASPET Abstracts 2011: Undergraduate Students

Periadolescent activation of the CB1 cannabinoid receptor prevents the normative developmental facilitation of
parvalbumin-positive interneurons in the prefrontal cortex
Accoh P, Simak N, Tseng KY
Department of Cellular and Molecular Pharmacology, RFUMS / The Chicago Medical School, North Chicago, IL 60064

Recent epidemiological findings indicate that cannabis use during adolescence is associated with an increased risk for schizophrenia and other psychiatric disorders. However, the neuronal basis underlying the association of cannabis use and psychiatric disorders remains unclear. Decreased levels of interneuron’s markers have been repeatedly observed in the prefrontal cortex (PFC) of subjects with schizophrenia. Among cortical interneurons, the subset expressing the protein parvalbumin (PV) are known to inhibit pyramidal neuron firing in the PFC. In addition, PV immunoreactivity is known to correlate to interneuron activity. Here we assessed the effect of repeated administration of the CB1 agonist WIN on interneuron function by measuring PV immunoreactivity in the PFC. We observed that PV immunoreactivity in the normal PFC follows a distinctive developmental trajectory: lowest in juveniles and maximal in adulthood. Repeated administration of WIN during adolescence prevented the developmental enhancement of PV immunoreactivity in the PFC. In contrast, PV staining in the adult PFC was unaffected by WIN exposure. These results indicate that excessive activation of CB1 receptors during adolescence prevents the normal developmental maturation of PV interneuron function in the PFC. We therefore hypothesized that activation of the CB1 cannabinoid receptor during adolescence could alter the developmental maturation of PFC interneurons, a developmental that trigger a disinhibited PFC state and contribute to an increased risk for psychiatric disorders. Supported by NIMH R01 MH086507 (KYT).

Jennifer Haick, Marla Hady, Robin Pals-Rylaarsdam
Benedictine University, Lisle, IL 60532

Heterotrimeric G-proteins cycle between an active/GTP-bound conformation and an inactive/GDP-bound conformation. Reduction in GTP hydrolysis by the G-protein alpha subunit causes constitutive activity of the protein. McCune-Albright Syndrome (MAS) is a disorder caused by a mutation that inhibits GTP hydrolysis in Gsα. Previous work from our laboratory identified an intragenic suppressor of the MAS mutation, which substituted two residues in the GTP-binding site. We have expressed this suppressor allele of Gsα, as well as wild-type (WT) and the MAS alleles in E. coli, partially purified them with affinity chromatography, and measured the ability of the isoforms of Gsα to bind and hydrolyze GTP. WT Gsα both binds and hydrolyzes GTP. The MAS allele can bind GTP well, but hydrolyzes it at a significantly lower rate than the WT protein. The suppressor allele is able to bind GTP, and exhibits an intermediate rate of GTP hydrolysis, suggesting that its suppression of the MAS mutation is due to partial restoration of GTPase function in the GTP binding site.

Daniela Janevska, Eraj Din, Robin Pals-Rylaarsdam
Benedictine University, Lisle, IL 60532

Heterotrimeric G-proteins cycle between an active/GTP-bound conformation and an inactive/GDP-bound conformation. McCune-Albright Syndrome (MAS) is a genetic disorder caused by a mutation (R201H) that inhibits GTP hydrolysis in Gsα, permanently activating the protein. We have developed a yeast model system for MAS in which mutations in the yeast G  subunit that mimic MAS (R297H) preven t colony formation. Previous students in the laboratory have used this system to identify an intragenic suppressor of the MAS mutation (L319P/D320V). In this presentation, we will describe our construction of a library of mutations in the yeast G subunit gene that will allow us to identify other suppressors of the MAS mutation using this yeast model. A library of 32,000 unique mutations in the Gα gene was constructed, the DNA purified, and preliminary screening the library for new intragenic suppressors of the MAS mutation has begun. Two new suppressor alleles have been identified. This work will allow future studies to thoroughly map the surface of Gα for sites which can inactivate the MAS allele, driving rational drug discovery of better treatments for MAS patients.

Garrick Moll, Joy Holowicki, Allison Wilson
Cell Biology/Toxicology, Benedictine University, Lisle, IL 60532

Cadmium has been identified as xenoestrogenic. Cadmium is a metal, yet it has the ability to bind to estrogen receptors. Estrogen is known to induce apoptosis within osteoclasts, which are the bone resorptive cells. Previous studies have shown that cadmium binds to estrogen receptor alpha (ER-α); it is postulated that the binding of cadmium to ER-α located on cellular surfaces inhibits the effect of estrogen on osteoclasts through a nongenomic pathway. Thus, cadmium could lead to premature bone loss and may exacerbate osteoporosis. Osteoclasts differentiated from RAW 264.7 cells and MitoCapture™ assay were used to confirm the findings of previous research that used DAPI/Phallodin staining. The analysis should inform that cadmium acts antagonistically with respect to estrogen, and that cadmium extends the viability of osteoclasts in the presence of estrogen. The second experiment intends to investigate the role of G-protein coupled estrogen receptor (GPER), a third estrogen receptor that also mediates nongenomic pathways in cells. Apoptosis after 1 hour was quantified with MitoCapture™. These tests will help clarify the mechanism of cadmium action in suppressing apoptosis in osteoclasts.

Cigarette smoke induces a pulmonary inflammatory response which leads to an increase activity of Mixed Lineage
Kinase-3 and possibly cell death
Arnav Rana, Velusamy Rangasamy, Gautam Sondarva, Joanna Bakowska and Suneet Mehrotra
Department of Molecular pharmacology and Therapeutics, Loyola University Medical Center, Maywood, IL 60153

Smoking is a central factor in many pathological conditions. Its role in neoplasm, lung and cardiovascular diseases has been well established for years. The mechanism of cigarette smoke toxicity is due to nicotine-enriched aerosol which is inhaled by smokers. The particle size of this aerosol is in micron-range, permitting efficient alveolar deposition and rapid absorption in the systemic blood. However, it is less acknowledged that cigarette smoking affects peripheral blood cell signaling. Cigarette smoke was shown to augment the production of numerous pro-inflammatory cytokines such as TNF-α, IL-1, IL-6, IL-8, GM-CSF and decreases the levels of anti-inflammatory cytokines such as IL-10. In our laboratory we have previously reported that increase in pro-inflammatory cytokine TNF-α, leads to an increase in kinase activity of Mixed Lineage Kinase-3 (MLK-3) a MAP3K member. We and others have reported that increase in MLK-3 kinase activity leads to activation of Jun N-terminal kinase (JNK) pathway, leading to cell death. Thus, we had carried out this study to elucidate the activity of MLK-3 in peripheral blood of smokers and non-smokers. Our results clearly demonstrate that smokers have augmentation in the kinase activity for MLK3, when compared to non-smokers. These results collectively suggest that smokers’ peripheral blood cells are more prone to cell death via activation of MLK3/JNK pathway.

Keith Solvang and Shubhik DebBurman
Biology Department, Lake Forest College, Illinois 60045

Parkinson’s disease (PD) is an incurable neurodegenerative disorder that affects four million people worldwide. Pathogenesis of PD includes aggregation of the protein α-synuclein into structures called Lewy bodies within dying midbrain dopaminergic neurons. α-Synuclein is also post-translationally modified through several mechanisms. Many neurodegenerative diseases, such as Alzheimer’s disease and Huntington disease, are linked to the imbalance of posttranslational modifications. Thus, a major hypothesis in PD is that the disproportionate levels of specific posttranslational modifications may contribute to those α-synuclein properties that make it toxic. Increasing evidence suggests phosphorylation and nitration are the most significant pathology-related modifications on α-synuclein. Three α-synuclein characteristics commonly linked to its toxicity include its ability to accumulate, to aggregate and to bind membrane phospholipids. However, the link between phosphorylation, nitration and these three properties are still not well understood in organismal models. Our lab has recently developed two yeast models that each recapitulates distinct aspects of these α-synuclein properties. In particular, budding yeast (Saccharomyces cerevisiae) models membrane association and fission yeast (Schizosaccharomyces pombe) models aggregation. Therefore, using both these yeasts, we evaluated the relationship between the pathological characteristics of α-synuclein and serine phosphorylation (S87, S129) and tyrosine nitration (Y39, 125, Y133, Y133). Specifically, we evaluated α-synuclein concentration (via Western blotting), aggregation and membrane association (via live cell e-GFP imaging) and cellular toxicity (via serial dilution colony growth in solid plates), and asked if these properties were altered when we blocked or constitutively activated the phosphorylation or nitration at each relevant site. We report three significant findings here. First, α-synuclein is phosphorylated in budding yeast. Secondly, constitutive phosphorylation decreased the ability to bind membrane phospholipids, however, blocking phosphorylation decreased aggregation and increased toxicity. Finally, nitration status affected membrane association in budding yeast and was toxic in fission yeast. This report provides multiple lines of evidence that post-translational modifications affect several PD-associated properties of α-synuclein. Supported by NIH R15 048508-02, NSF MRI 0115919, NSF CCLI 0310627.

Dahlia Sultan, Raquel Tobar-Rubin*, Robin Pals-Rylaarsdam
Benedictine University, Lisle, IL 60532

Heterotrimeric G-proteins cycle between an active/GTP-bound conformation and an inactive/GDP-bound conformation. Reduction in GTP hydrolysis by the G-protein alpha subunit causes constitutive activity of the protein. McCune-Albright Syndrome (MAS) is a genetic disorder caused by a mutation that inhibits GTP hydrolysis in Gsα. Biopsies from MAS patients show R201H, R201S, and R201C mutations. Previous work modeled the MAS mutation in a yeast system, and identified an intragenic suppressor of the MAS mutation, which substituted two residues in the GTPbinding site. We characterized the ability of mutations at F222 and D223 in human Gsα to suppress the constitutive activity of the protein caused by mutations at R201 found in MAS patients. Site-directed mutagenesis introduced mutations into Gs at residues F222 and D223. Following expression in mammalian cells; basal and receptor-mediated cAMP production was measured by ELISA. The original suppressor, F222P/D223V, reduced basal cAMP levels compared with the R201H allele and signaled through G-protein mediated pathways. F222P and D223V alone each suppressed the constitutive activity of the R201H allele. The F222P/D223V mutation was able to suppress the constitutive activity of a serine substitution at R201. Finally, Substitution of D223 with nonpolar (A), but not acidic (E) or polar (N) residues was able to suppress the R201H constitutive activity similarly to the D223V substitution.

Last Updated: August 3, 2017

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