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Microfluidic Oxygen Control Demonstrates Crosstalk Between Normoxic and Hypoxic Stem Cells
Megan L. Rexius, David T. Eddington, (University of Illinois at Chicago, Department of Bioengineering) and Jalees Rehman (University of Illinois at Chicago, Department of Pharmacology)
Hypoxia regulates adult and embryonic stem cell function as well as vascular differentiation and regeneration. Homogenous hypoxia is known to increase secretion of pro-angiogenic vascular endothelial growth factor (VEGF) expression in human mesenchymal stem cells (MSCs) which can activate angiogenesis in endothelial cells. Current approaches which use homogenous oxygen levels to study hypoxia in cells and tissues do not replicate physiological oxygen gradients formed as a result of oxygen diffusion from the microvasculature. Furthermore, traditional hypoxia chambers do not allow assessment of real-time paracrine crosstalk which naturally occurs in vivo between cells exposed to varying oxygen levels. The objective of this study was to investigate the effect of close proximity interactions between normoxic and hypoxic cells.
We have developed an open-well microfluidic device with precise control of oxygen gradients. Our cell culture platform allows different oxygen levels to be selectively applied to distinct cell types in a co-culture (e.g. one cell type can be hypoxic while the other is normoxic), while permitting paracrine interactions between the distinct cell types via shared cell culture media. We present for the first time hypoxic upregulation of VEGF in MSCs suppressed in the presence of close-proximity normoxic MSCs, when compared to hypoxic MSCs without adjacent normoxic cells. Similarly, in co-cultures of hypoxic MSCs and normoxic microvascular endothelial cells (MVECs), normoxic endothelial cells also exerted a mitigating effect on VEGF expression. These findings suggest that when cells are exposed to an oxygen landscape with varying oxygen levels, neighboring normoxic cells may modulate cellular hypoxia responses in MSCs. However, this modulating effect does not appear to be a global response and does not impact metabolic genes such as the glucose transporter (Glut1).
Our microfluidic cell culture platform overcomes the limitations of current technology in order to study paracrine interactions across an oxygen gradient. Continuation of this research aims to manipulate the outcomes of hypoxic signaling. We also anticipate that the research results will lead to a better understanding of how to control angiogenesis and improve chronic ischemic disorders. (Back to top)
Multipotent stromal cells in micromass culture for studying mechanisms of antifibrotic drugs
Chan DD, Li J, Gorski DJ, DelaMotte CA, Predescu DN, Plaas AHK (Rush University and Cleveland Clinic)
TGF-β is a major regulator of numerous cellular processes following soft tissue injury, including proliferation of granulation tissue stromal cells, contraction, and regeneration of collagenous matrices. Dysregulation of TGF-β1 responses during healing can lead to fibrosis, hypertrophic scarring, or contractures. Following cartilage damage in the knee, wild-type mice show a robust healing response, whereas mice lacking hyaluronan synthase 1 (Has1KO) develop extensive soft tissue fibrosis, chronic inflammation, and severe osteoarthritis. Treatment of Has1KO mice with the antifibrotic Pirfenidone reduced inflammation and prevented cartilage and bone damage but only minimally decreased the fibrosis. Since our previous studies showed that chronic intra-articular TGF-β1 application resulted in stromal cell proliferation and fibrotic remodeling, we examined the effect Pirfenidone on TGFb1 responses of stromal cultures from wild-type and Has1KO mice. Cells were isolated from abdominal adipose and passaged 3-times. For micromass cultures, cell suspensions (5×107 cells/mL AMEM/10%-FCS/ascorbate) were dispensed as 20-µL drops, with fresh medium added following adherence and aggregation. After 24h, media was replaced and supplemented with TGF-β1, Pirfenidone and/or TGFβRI/II-inhibitor. Media and cell layers were harvested after 24h for QPCR of Col1a1, Col3a1, Has1, and Has2 mRNA levels and Western blotting for type-1 collagen. In wild-type cells, TGF-β1 increased Col3a1 (~3-fold) and Has1&2 (~4-fold) mRNA levels with no significant changes to Col1a1. Pirfenidone attenuated the increase in Col3a1 mRNA, but had no effect on stimulated levels of Has1&2. TGFβRI/II-inhibitor prevented all TGF-β1-induced effects. In Has1KO cells, TGF-β1 increased Col1a1 mRNA (~5-fold), with no effect on Col3a1 or Has2. Moreover, Pirfenidone and TGFβRI/II-inhibitor only minimally decreased TGF-β1 effects in Has1KO stromal cultures, suggesting the activation of alternative profibrotic signaling pathways, to modulate collagen gene expression in the absence of HAS1. This distinction between phenotypes was also seen when cultures were assayed for collagen-1 production using Western blotting. With Pirfenidone, collagen-1 secretion was reduced in wild-type cells but remained unchanged in Has1KO cells. In conclusion, the use of multipotent stromal cells in micromass cultures is a useful tool to study the effect of antifibrotics and additionally discover new genes in the fibrosis pathways and their sensitivity to such drugs. (Back to top)
Human neurons derived from induced pluripotent stem cells model chemotherapeutic induced peripheral neuropathy
Heather E. Wheeler, Claudia Wing, Shannon M. Delaney, Masaaki Komatsu, and M. Eileen Dolan, (Section of Hematology/Oncology, Department of Medicine, University of Chicago)
Chemotherapy-induced peripheral neuropathy (CIPN) is the major dose-limiting toxicity of many anti-cancer drugs. The mechanisms underlying CIPN have not been precisely determined and few human neuronal models to study CIPN exist. We describe the development and feasibility of using human neurons derived from induced pluripotent stem cells (iPSCs) as a genetically diverse model for CIPN. Upon treatment of human neurons with the neurotoxic chemotherapeutic paclitaxel, vincristine or cisplatin, we identified reproducible decreases in neurite outgrowth phenotypes. Distinct morphological changes in damage among the drugs reflect differences in their mechanisms of action and clinical manifestations of CIPN. We show the potential of the model for gene perturbation functional studies by optimizing conditions for siRNA transfection. Upon reprogramming four unrelated lymphoblastoid cell lines (LCLs) into iPSCs and differentiating them into neurons, we demonstrate that the variance in several neurite outgrowth phenotypes upon treatment with one of the neurotoxic drugs is greater between than within individuals, demonstrating the potential of this model for larger genetic association studies. Genetically diverse cell types derived from iPSCs relevant to tissues of drug toxicity have the potential to greatly impact the field of pharmacogenomics. We present key chemotherapeutic-induced phenotypes in human iPSC-derived neurons that model CIPN. The human neuron model will allow both for functional studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN. (Back to top)
Disease Modeling Using Patient Specific iPSC
Galat Yekaterina, Irina Elcheva, Mariana Perepitchka, Vasiliy Galat
Defining the earliest phenotypic and molecular signs of abnormal development in patients with various mutations and chromosomal abnormalities will not only contribute to the overall understanding of these syndromes, but also facilitate the development of new treatments that could prevent or lower the risk of congenital and sporadic disorders. Our research, as well as others’, has shown that experimental models of differentiation from pluripotent stem cells (PSCs), both embryonic (ESCs) and induced pluripotent stem cells (iPSCs), recapitulate major steps of the development in embryo. Our lab focuses on deriving iPSC from patients with various disorders and utilizing the method of in vitro differentiation to broaden our understanding of such prominent disorders as Down Syndrome, as well as, increase our knowledge of rare neurobehavioral disorders caused by mutations in RAI1 and GRIN2B genes.
Down syndrome (DS) is a complex disease caused by a trisomy of human chromosome 21 (HSA21) that occurs at a rate of 1 in every 750 live births. Children with DS show a spectrum of clinical anomalies implicating the development of all embryonic lineages. Our lab works to understand how HSA21 affects formation of mesodermal lineages, i.e. mesenchymal, endothelial, cardiac, and blood progenitors on molecular and cellular levels. We anticipate that at least some phenotypic malformations manifested in musculoskeletal, hemato-endothelial, and cardiovascular tissues of DS can be modulated at the mesodermal precursor stage of development.
Additionally, successfully generated and characterized a novel patient specific iPSC model for RAI1 and GRIN2B mutations both of which manifest in a complex phenotype, including intellectual disability. Mutation in the RAI1 gene is characterized by multiple congenital defects including intellectual disabilities, sleep disturbances, craniofacial and skeletal disorders. Several recent studies have identified association of mutations in the GRIN2B gene with autism, schizophrenia and epilepsy. This model provides a unique opportunity to induce neuronal differentiation in stem cells from siblings that differ in single dominant mutations critical to normal neuronal differentiation. The model was established using a non-integrating Sendai virus reprogramming system. The generated iPSCs display embryonic stem cell-like morphology, express stem cell markers, and are capable of differentiation to three germ lineages. We further differentiated the iPSC lines to neural progenitors. We believe that by comparing gene expression data from mutant iPSCs, to ”control” iPSCs and their corresponding neuronal derivatives it will be possible to discover mutation related alterations that can then be linked to impaired human neuronal development and provide a basis for discovery of novel therapeutic targets.
In summary, iPSC offer an invaluable disease modeling system, as well as, drug screening platform by providing the type of cells that are not attainable directly from patients. (Back to top)
A 3D High-Content/High-Throughput Screening Platform to Enhance Multilineage Stem Cell Differentiation and Improve Human Pancreatic Islet Viability
Amit Paul, Enza Marchese, David Franz, Sumaira Yahya, Kirstie Danielson, Jose Oberholzer, and Michael Cho (Department of Bioengineering, University of Illinois at Chicago and Division of Transplant Surgery, University of Illinois at Chicago)
The cytoskeleton of human mesenchymal stem cells (MSCs) alters dramatically during differentiation. These lineage-dependent changes cause MSCs to exhibit unique cytoskeletal “fingerprints” along the various stages of differentiation. To investigate the relationship between these dynamic cytoskeletal configurations and MSC lineage commitment on a large scale, we created a novel computational algorithm that integrates high-content screening (HCS, detailed analysis of single cells) and high-throughput screening (HTS, rapid/high-volume experimentation). Most notably, the entire HCS/HTS process is performed in an automated environment using a custom-engineered 3D cell/tissue imaging system. While undergoing multilineage differentiation (adipogenesis, osteogenesis, chondrogenesis, myogenesis, neurogenesis, vasculogenesis, and hepatogenesis), MSCs were fluorescently labeled at crucial differentiation time points for cytoskeletal and lineage-specific markers. All samples were spatially-resolved/scanned/imaged via high-resolution (100-200nm) 3D confocal/multiphoton laser scanning microscopy. This HCS/HTS system was integrated with laser capture microdissection to automatically identify and capture differentiating stem cells in a selective manner for gene expression profiling. Additionally, the dynamic lineage-dependent biomechanical changes were correlated using atomic force microscopy. With the abundance of quantitative experimental data, we developed a sophisticated cytoskeletal fingerprinting process that accurately analyzes & predicts the mechanical transformations which are potentially required to initiate the onset of stem cell differentiation into multiple lineages. With the assistance of this advanced HCS/HTS platform, many aspects of MSC differentiation were thoroughly elucidated. Perhaps most significant was a consistent pattern observed in all types of differentiation: the cytoskeleton remodeled significantly before lineage-specific cellular changes occurred. This strongly suggests that cellular mechanical transformations are a precursor to stem cell differentiation. The combinatorial approach of 3D high-resolution imaging and computational modeling provides a robust system to better understand MSC differentiation and ultimately reduce heterogeneity in multipotent/pluripotent stem cell populations. This 3D HCS/HTS imaging system has also been applied to assessing isolated human pancreatic islets from cadaveric donors (large/thick multicellular structures) for their active beta-cell/alpha-cell/delta-cell content. We are utilizing the results of this analysis to engineer superior methods of improving human islet survival/viability for clinical transplantation. (Back to top)
Endothelin ETA Receptor Antagonist Reverses Naloxone-precipitated Opioid Withdrawal in Mice
Shaifali Bhalla, Melissa Tapia, Gwendolyn Pais, and Anil Gulati (Chicago College of Pharmacy and College of Health Sciences, Midwestern University, IL)
Background: The long term use of opioid analgesics for the treatment of pain results in the rapid development of tolerance and dependence leading to severe withdrawal symptoms. Several neurotransmitter mechanisms have been proposed to play a role in the actions of opioid analgesia and withdrawal. We have previously demonstrated that endothelin-A (ETA) receptor antagonists potentiate morphine and oxycodone analgesia in rodents. We also demonstrated that ETA receptor antagonists eliminated tolerance by restoring the antinociceptive responses to both morphine and oxycodone in opioid tolerant animals.
Objective: The present study was designed to investigate the involvement of central endothelin mechanisms in opioid withdrawal. The effect of intracerebroventricular administration of ETA receptor antagonist, BQ123, on morphine and oxycodone withdrawal was determined in male Swiss Webster mice.
Methods: Opioid tolerance was induced by twice-daily injections of morphine for three days, and once-daily injections of oxycodone for five days. Withdrawal was precipitated by opioid receptor antagonist, naloxone, on day 4 for morphine studies and day 6 for oxycodone studies. Expression of ETA receptors, ETB receptors, VEGF and NGF was determined using Western Blotting technique.
Results: Pretreatment with BQ123 reversed the hypothermia and loss of body weight in mice undergoing morphine and oxycodone withdrawal. BQ123 also significantly reduced the number of wet shakes, rearing and jumping behavior during withdrawal. Western blotting studies indicated no changes in the expression of VEGF, ETA receptors, and ETB receptors following administration of vehicle or BQ123 in the brain. Although statistically insignificant, we observed a slight tendency toward decrease in ETA receptor expression in BQ123-treated animals in control and withdrawal groups. NGF expression was not affected in morphine withdrawal but significantly decreased in the brain during oxycodone withdrawal. Expression of NGF was not altered by BQ123 pretreatment.
Conclusion: These studies are the first to demonstrate that ETA receptor antagonists not only eliminate antinociceptive tolerance but also reverse withdrawal symptoms of morphine and oxycodone. These findings support the hypothesis that ETA receptor antagonists in combination with opioid analgesics provide adequate analgesic response without the addiction potential and withdrawal symptoms of opioids. (Back to top)
The Marine Pharmacology and Pharmaceuticals Pipeline in 2014
A.M.S. Mayer, Dom Dop Van Nguyen and K.B.Glaser (Department of Pharmacology, CCOM, Chicago College of Pharmacy, Midwestern University, IL and AbbVie Inc.)
The status of the marine pharmacology and pharmaceutical pipelines was assessed in early 2014. There were five FDA-approved marine-derived drugs in the US market, namely cytarabine for cancer (Cytosar-U®, Depocyt®, FDA-approved 1969), ziconotide for pain (Prialt®, FDA-approved 2004), omega-3-acid ethyl esters for hypertriglyceridemia (Lovaza®, FDA-approved 2004), eribulin mesylate for cancer (Halaven®, FDA-approved 2010), and brentuximab vedotin for cancer (Adcertis®, FDA-approved 2011), while vidarabine as an antiviral (Vira-A®, FDA-approved 1976) was no longer available, and trabectedin for cancer (Yondelis®, FDA-orphan drug approval 2005) being EU-registered. In early 2014, the clinical marine pharmaceutical pipeline (Reviewed in Mayer et al. TIPS 31:255-265, 2010) consisted of 10 marine-derived compounds in clinical development. Included in the clinical marine pharmaceutical pipeline were three new monoclonal antibodies conjugated to monomethyl auristatin E, a synthetic analog of the marine compound dolastatin, which were in either Phase I, Phase II or Phase III clinical trials. Of note is that there are currently at least 14 other auristatin-containing antibody drug conjugates in clinical development. Updated information on the clinical marine pharmaceutical pipeline is available at http://marinepharmacology.midwestern.edu/clinPipeline.htm. Furthermore, the global preclinical marine pharmacology pipeline reported multiple marine chemicals with novel mechanisms of action (Reviewed in Mayer et al. MARINE DRUGS 11:2510-2573, 2013). Thus in early 2014, both the marine pharmacology preclinical pipeline as well as the marine clinical pharmaceutical pipeline remained very active. Supported by Midwestern University. (Back to top)
Effects of Metformin on Bethanechol-Induced Contractions of the Lower Esophageal Sphincter of the Rat
Jacob D. Peuler, Laura E. Phelps and Patrick W. Murphy (CCOM and Midwestern University)
Metformin is widely used in the treatment of diabetes. We have shown 1) that at millimolar concentrations it can markedly inhibit contractions of the isolated rat bladder as induced by the muscarinic receptor agonist bethanechol and 2) that this effect can be antagonized by the potassium (K) channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4AP) but not barium or glyburide (The FASEB Journal 2012 ; 26 :1049.2). Thus, metformin may be opening some but not other subpopulations of K channels known to exist in smooth muscle cell membranes of the bladder wall. We tested for these same effects in the lower esophageal sphincter of the rat because 1) by contracting this tissue bethanechol is used to treat gastroesophageal reflux disease (which is common in diabetic patients) and 2) metformin is known to reach millimolar concentrations in gastrointestinal tissues (including the esophagus) after standard oral dosing. Our results were the same as we observed previously in rat bladder. At 2-3 minutes after its administration, 5 millimolar metformin inhibited bethanechol’s contraction of the isolated esophageal sphincter by 61±3% in the absence of K channel blockers (control). In the presence of TEA and 4AP, it only inhibited that contraction by 18±4% and 28±5%, respectively (p
Patrick W. Murphy was supported by the Midwestern University Summer Fellowship Program. Laura E. Phelps and Jacob D. Peuler were supported by Midwestern University intramural funds. (Back to top)
Polybrominated Diphenyl Ether Congener 47 Increases Aldosterone Secretion in a Human Adrenocortical Cell Line
BM Dungar and PG Kopf. (Department of Pharmacology, Midwestern University, IL)
Elevated circulating aldosterone levels are associated with hypertension, thrombosis formation, cardiac hypertrophy, and congestive heart failure. Polybrominated diphenyl ethers (PBDEs) and their metabolites have been previously shown to alter various endocrine biosynthetic pathways including thyroid hormone, estrogens, and androgens. Additionally, PBDEs have been shown to accumulate in the adrenal gland. Therefore, we characterized the effect of BDE-47 on aldosterone secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or various concentrations of BDE-47 (10 nM-100 µM). After 72 h, cell viability, aldosterone secretion, and gene expression of enzymes and cofactors involved in aldosterone synthesis was examined. BDE-47 decreased cell proliferation at 10 and 100 µM. BDE-47 induced basal aldosterone secretion at 10 and 100 µM. BDE-47 increased ACTH-stimulated aldosterone secretion at 1, 10, and 100 µM. BDE-47 also increased Ang II-stimulated aldosterone secretion at 10 µM, but decreased Ang II-stimulated aldosterone secretion at 100 µM. Gene expression of several enzymes and cofactors involved in aldosterone synthesis were increased by 10 µM BDE-47. These data indicate that BDE-47 disrupts the regulation of aldosterone secretion and provides further evidence that PBDEs are potential endocrine disruptors. (Back to top)
Preclinical Evaluation of Cystatin C as an Early Biomarker of Cadmium Nephrotoxicity
Walter C. Prozialeck, Aaron Van Dreel, Christopher D. Ackerman, Ian Stock, Alexander Papaeliou, Joshua R. Edwards, Peter C. Lamar, Karyn DiNovo and Vishal S. Vaidya (Department of Pharmacology and Department of Physiology, Midwestern University, IL and Renal Division, Brigham and Woman’s Hospital, Harvard Medical School, MA)
Chronic exposure to cadmium (Cd) results in injury to the proximal tubule that is characterized by polyuria and proteinuria. As a result of the widespread use of Cd in industry and its extensive dissemination in the environment, there has been considerable interest in the identification of early biomarkers of Cd-induced kidney injury. Cystatin C is a low molecular weight protein (13.3 kilodaltons) that has been proposed as a sensitive biomarker for renal injury. Under normal circumstances, cystatin C is readily filtered at the glomerulus and then taken up and metabolized by epithelial cells of proximal tubule. The objective of the present study was to determine if cystatin C might be a useful early biomarker of Cd nephrotoxicity. Male Sprague-Dawley rats were given daily injections of Cd (0.6 mg/kg, 5 day per week) for up to 12 weeks. At 6, 9 and 12 weeks, 24 hour urine samples were collected and analyzed for levels of cystatin C, total protein, creatinine and kidney injury molecule-1 (Kim-1). Blood samples were collected and assayed for creatinine and cystatin C. Representative animals were euthanized and their kidneys were processed for histopathologic analyses and the immunohistochemical visualization of cystatin C and other molecules of interest. The results showed that Cd caused a significant (1-2 fold) increase in urinary excretion of cystatin C that was evident at all time points examined. Urinary Kim-1 showed even more pronounced elevations at the same time points. No change in urinary protein was evident until 12 weeks, whereas urinary creatinine was not affected by Cd. Serum levels of creatinine and cystatin C were not altered by cadmium. Histochemical analyses revealed evidence of proximal tubule injury (separation and retraction of cells, with little evidence of overt necrosis and only low levels of apoptosis). Immunolabeling studies showed that Cd caused marked changes in the pattern of cystatin C distribution in proximal tubule. Control samples showed a speckled pattern of labeling just beneath the apical (brush border) surface of the epithelial cells. By contrast, the samples from Cd treated animals exhibited diffuse cystatin labeling in the cytoplasm, on the cell surface and in the lumen of the tubules. Both the general pattern of labeling and the Cd-induced changes for cystatin C paralleled those of the brush border transport protein megalin, which has been implicated as a mediator of cystatin C uptake in the proximal tubule. These results indicate that Cd increases the urinary excretion of cystatin C, and they suggest that this effect may involve disruption of megalin-mediated uptake of cystatin C by epithelial cells of the proximal tubule. (Back to top)
Intersectin-1s Downregulation Enhances Lung Cancer Metastasis
Niranjan Jeganathan, Fei Sha, Monal Patel, Christina Bardita, Stephen Wood, Dan Predescu, Sanda Predescu (Rush University Medical Center, IL, Pulmonary and Critical Care Medicine, Pharmacology and Immunology)
Lung cancer is the leading cause of malignancy-related mortality worldwide. Approximately 90% of cancer-related deaths are the result of metastasis. A single therapeutic approach or targeting individual pathways have not significantly improved survival due to the complicated nature of the disease. Intersectin-1s (ITSN), a membrane bound multidomain scaffold protein, is emerging as a key regulator in a number of cell signaling pathways involved in tumorigenesis (EGFR. Ras, PI3K). ITSN has been shown to be significantly downregulated in many solid organ malignancies, especially lung cancer which was confirmed in our lab using lung cancer cell lines and human lung cancer tissues. Additionally, our findings indicate that changes in the level of ITSN expression induce significant alterations to cytoskeleton structures (actin, vimentin) and focal adhesion proteins (vinculin, paxillin). These morphological changes lead to epithelial mesenchymal transition (EMT) which is the hallmark of cancer metastasis.
To investigate ITSN’s role in lung cancer, A549 cells (adenocarcinoma human lung cancer cells) and A549 cells with ITSN expression restored (using myc-ITSN plasmid) were studied using biochemical and morphological approaches. Restoration of ITSN expression resulted in conversion of small/thin filaments of actin into thick actin bundles, increased number of vinculin adhesion particles, increased actin/vinculin co-localization and decreased paxillin expression. ITSN restoration decreased phosphorylation of vimentin and induced collapse of the vimentin filament network. These changes favor inhibition of EMT which was supported by the wound healing assay with time-lapse microscopy (41% inhibition in cell migration noted with restoration of ITSN expression). Additional investigation showed that ITSN inhibits formation of SOS1-EPS8 complex and thereby inhibits Rac1 activation. Together, these results suggest that ITSN downregulation in lung cancer leads to increased Rac1 activation which alters the cytoskeleton structures in favor of EMT. Restoration of ITSN expression inhibits this pathway and could be a potential therapeutic option. (Back to top)
Gap Junction Protein Connexin 43 (Cx43) Functions to Exacerbate Vascular Permeability
James J. O’Donnell III, Anna A. Birukova, Eric C. Beyer, Konstantin G. Birukov (Department of Medicine, Department of Pediatrics, University of Chicago, IL)
Increased vascular permeability is a hallmark of inflammatory diseases such as acute lung injury (ALI) and sepsis, which account for approximately 75,000 and 300,000 deaths per year in the United States, respectively. Emerging evidence suggests that connexins, the building blocks of dihexameric intercellular gap junctions, may regulate vascular permeability as well as other indices of inflammatory signaling. We investigated the potential impact of connexins on vascular permeability in lung endothelium by testing whether ALI-associated inflammatory stimuli altered connexin expression and function, and then assessing the effects of altered connexin expression on permeability through loss-of-function studies. Thrombin, lipopolysaccharide (LPS), and cyclic stretch all upregulated connexin 43 (Cx43) mRNA and protein expression. Cx43 was localized at the junctional membranes and around the nuclei in untreated as well as LPS- or thrombin-treated cells. Dye transfer assays showed that both LPS and thrombin treatment enhanced intercellular gap junction communication, demonstrating that upregulated Cx43 was functional. Inhibition of gap junctions with carbenoxolone attenuated the thrombin-induced decrease in transendothelial resistance, an index of permeability. Carbenoxolone also inhibits thrombin-induced phosphorylation of myosin light chain (MLC,) the primary mechanism of thrombin-induced permeability. Cx43 knockdown attenuated thrombin- and LPS-induced permeability as measured by transendothelial resistance, and Cx43 knockdown also abrogated thrombin-induced MLC phosphorylation. As Cx43 loss-of-function attenuated thrombin- and LPS-induced permeability, Cx43 functions to exacerbate permeability. Overall, our data suggest that increased Cx43 under inflammatory conditions may exacerbate vascular permeability, and lays the groundwork for a role of gap junctional intercellular communication in the development of ALI and sepsis. (Back to top)
Carbon-Hydrogen bond functionalization using novel artificial metalloenzymes for enhanced selectivity
Lee A. Solomon, Chen Zhang, Jared C. Lewis (Department of Chemistry, University of Chicago, IL)
Carbon-Hydrogen (C-H) bonds are ubiquitous throughout biology and form the heart of many industries. Manipulating these bonds is a major factor in social and economic issues worldwide owing to the difficulty with the associated processes. Synthetic chemical methods often generate mixtures with poor site and enantiomeric selectivity. Biological methods, while avoiding issues with selectivity, lack the scope to functionalize C-H bonds in ways useful to pharmaceutical and energy industries. We bridge this gap using a novel hybrid of the two systems: artificial metalloenzymes (ArM’s). ArM’s utilize protein architecture to bind and orient small molecules in order to react with an attached synthetic cofactor. The specificity of the substrate-binding site does not allow for racemic mixtures of products nor extraneous sites to be activated while the synthetic cofactors are capable of non-natural reactions on substrates both present and absent in biology. We have designed an ArM, covalently linking a Mn-terpyridine cofactor to the protein Nitrobindin, capable of converting a styrene group to an epoxide with minor enantiomeric selectivity. Building off this initial activity, we are beginning to enhance the yield and selectivity of this reaction through a combination of rational design and directed evolution mutations, generating other interactions to hold other substrates in a specific orientation. We are also aiming to broaden the substrate scope to incorporate other drug precursors providing the pharmaceutical industry with novel strategies for drug generation for less money and higher yield. (Back to top)
Effects of neuronal nitric oxide synthase (nNOS) inhibition on L-DOPA-induced striatal ERK1/2 phosphorylation in the 6-OHDA rat model of Parkinson's disease
Fernando E. Padovan-Neto and Elaine A. Del Bel (Department of Neuroscience, the Chicago Medical School at RFUMS, North Chicago, IL and Department of Behavioral Neuroscience, FMRP, University of São Paulo, Ribeirao Preto, SP, Brazil)
The role of the atypical neurotransmitter nitric oxide (NO) in L-DOPA-induced dyskinesia (LID) was demonstrated by the anti-dyskinetic properties of NO synthase (NOS) inhibitors. A molecular marker of LID is the abnormally high levels of phosphorylated extracellular-regulated kinase 1/2 (p-ERK1/2) within the striatum. This study aimed to analyze the anti-dyskinetic effect of the neuronal NOS inhibitor 7-nitrondazole (7-NI) on neuronal NOS (nNOS) - soluble guanylyl cyclase (sGC) pathway and on the state of phosphorylation of ERK1/2 in the dorsal striatum. 6-OHDA-lesioned rats were divided into four treatment groups which received the combination of the following treatments (chronically, once a day, for 21 days, n=5-9/group): vehicle/saline, 7-NI/saline, vehicle/L-DOPA and 7-NI/L-DOPA. 7-NI (30 mg/kg) or its vehicle (2 ml/kg) was administered 30 min prior to L-DOPA (10 mg/kg) or saline (2 ml/kg). A group of Sham-operated animas (n=6) was also included. Rats were examined behaviorally for a rat dyskinesia scale and stepping test. Western blots were performed in total homogenates of the dorsal striatum to analyze the expression of nNOS, sGC and p-ERK1/2. Chronic L-DOPA reversed akinesia and induced the appearance of axial, limb, orofacial and locomotive dyskinesia in 6-OHDA-lesioned rats with >90% of dopaminergic cell loss in substantia nigra compacta. LID was associated with an up-regulation of nNOS, sGC and p-ERK proteins in the dorsal ipsilateral striatum. Prolonged administration of 7-NI blocked the manifestation of LID by chronic L-DOPA treatment without interfering with the antiparkinsonian effect of L-DOPA as demonstrated by the stepping test. Chronic 7-NI also inhibited L-DOPA-induced up-regulation of nNOS, sGC and p-ERK proteins in the dorsal ipsilateral striatum. The manipulation of NO system with nNOS inhibitors as an anti-dyskinetic prophylactic approach was effective in normalizing the up-regulation of nNOS, sGC and p-ERK1/2 protein expression in ipsilateral dorsal striatum. The anti-dyskinetic effects of NOS inhibitors in striatal ERK1/2 expression may contribute to the understanding of this newly non-dopaminergic therapy for the management of LID. (Back to top)
Novel Retrieval Receptor important for Toxoplasma gondii survival
Abhishek Tripathi and Kristin Hager (University of Notre Dame, IN and Loyola University of Chicago, IL)
Toxoplasma gondii is a eukaryotic pathogen possessing a remnant plastid called the apicoplast. T. gondii utilizes classical Endoplasmic Reticulum Deficient‐2 (ERD2) receptor for retrograde trafficking that aids in maintaining protein homeostasis. ERD2‐ like receptors were identified in Toxoplasma and their distribution restricted to plastid‐containing species; hence they are named Plastid Associated ERD2‐Like Proteins (PERLs). In this abstract we describe the cloning, localization, and characterization of one the T. gondii PERL homologues, TgPERL‐51 (TgP51). Protein trafficking to the apicoplast is still a matter of debate. Experiments with Golgi disruption or tagging of apicoplast proteins with an HDEL motif suggest that most of the apicoplast proteins are trafficking from the ER to the apicoplast via a compartment other than, or in addition to, the Golgi, even though apicoplast protein processing is Brefeldin‐A (BFA) sensitive step. In this trafficking scenario, an additional receptor could be functioning in non‐classical ER retrieval, and PERLs may perform this function. TgP51 possesses the unique bipartite sequence characteristic of some apicoplast targeted proteins and TgP51‐GFP/mCherry localization overlaps with the apicoplast region. Bioinformatics analysis also reveals a novel anchor domain in TgP51, a domain that until recently has only been associated with plastid‐targeted proteins in Euglena and dinoflagellates. We demonstrated that unlike TgERD2, TgP51 does not complement the yeast knockout. TgP51 localizes in BFA sensitive manner. We also show that knockout of TgP51 gene in Ku80cells is Lethal in a "delayed death" phenotype and knock out parasites display abnormal apicoplast morphology in comparison to controls. Lastly, we propose a model for probable role of PERL in retrieval mechanism and postulating that could be a potent future drug target or a vaccine candidate to control Apicomplexan infections in humans as well as in animals. (Back to top)
Late Adolescent Expression of GluN2B Transmission in the Prefrontal Cortex Is Input-Specific and Requires Postsynaptic Protein Kinase A and D1 Dopamine Receptor Signaling
Eden Flores-Barrera, Daniel R. Thomases, Li-Jun Heng, Daryn K. Cass, Adriana Caballero, and Kuei Y. Tseng (Department of Cellular and Molecular Pharmacology, the Chicago Medical School at RFUMS, North Chicago, IL)
Refinement of mature cognitive functions such as working memory and decision-making typically take place during adolescence. Although the acquisition of these functions is linked to the protracted development of the prefrontal cortex (PFC), the cellular mechanisms that support these changes remain elusive. In this regard, dopamine facilitation of NMDAR transmission has long been recognized to critically regulate PFC functions in adulthood. Here we found that only NMDA receptor transmission onto the apical dendrite of layer V pyramidal neurons undergoes late adolescent remodeling due to the emergence of NR2B function. Both PKA and D1R signaling are required for the functional expression of NR2B transmission to sustain PFC plasticity in response to ventral hippocampal, but not basolateral amygdala inputs. Thus, the late adolescent acquisition of NR2B function provides a mechanism for the maturation of dopamine action by enabling D1 receptor regulation of PFC responses in an input-specific manner. (Back to top)
Isolation and Analysis of Ngn3/CD133+ Endocrine Precursor Cells From Human Exocrine Tissue
James J. McGarrigle, Pilar Vaca-Sanchez, Enza Marchese, Sang Joon Ahn, Andre Thomas, Mike Shamblott, Jose Oberholzer (University of Illinois at Chicago, IL and University of South Florida, FL)
Type I Diabetes Mellitus (T1DM) is a metabolic disease caused by the autoimmune destruction of insulin-producing beta cells in human pancreatic islets. Human islet transplantation is now seen as a viable treatment against T1DM, however the procedure is limited due to the lack of availability of pancreata from human donors. At present the vast majority of donor pancreas tissue, mainly comprised of exocrine tissue, is discarded following the isolation of islet cells from the organ. During pancreatic development, cells that commit to an endocrine cell fate begin to express the basic Helix-Loop-Helix (bHLH) transcription factor Neurogenin 3 (Ngn3), which is the key factor that triggers their differentiation into all islet cell types, including the insulin producing beta-cells. Furthermore it has been shown that Ngn3 endocrine progenitor cells in the human pancreas co-express CD133, a transmembrane glycoprotein. Exploiting the fact that exocrine and endocrine pancreatic tissue have a common embryonic origin, we set about to isolate CD133+ progenitor cells, isolated from exocrine tissue discarded during human pancreatic islet isolations, which are also known to express Ngn3. Using Magnetic Cell Sorting, CD133+ cells were isolated and cultured in a short term suspension culture. These individual cells were shown to aggregate into sphere like structures, known as pancospheres. Histological analysis of the pancospheres indicated the presence of Pdx1, a transcription factor necessary for pancreatic development and beta-cell maturation, as well as the protein chromogranin A. Furthermore in vivo analysis of these pancospheres in nude mice indicated an alteration in blood glucose levels during intraperitoneal glucose tolerance test (IPGTT). Together these data indicate that endocrine precursor cells can be isolated from “waste” exocrine tissue following human pancreatic islet isolation and that such cells, when cultured, can develop into pancospheres that can express proteins necessary for pancreatic development and beta-cell maturation. Further in-depth genetic, proteomic and functional analysis of these pancopheres will have to be performed to determine their exact significance to the field of diabetic research. (Back to top)
5-HT1A receptors in the nucleus tractus solitarius facilitate ventilatory responses to hypoxic hypercapnia and sympathetic recovery following hypotensive hemorrhage
Jaime E. Vantrease, Nichole L. Dudek, Karie E. Scrogin (Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, IL)
The 5-HT1A receptor agonist, 8-OH-DPAT, improves recovery from hemorrhage by stimulating sympathetically-mediated venoconstriction and cardiac output. The sympathoexcitatory effect of 8-OH-DPAT is only observed in hypovolemic rats, and is attenuated by sinoaortic denervation. Lesion of serotonergic nerve terminals in the nucleus tractus solitarius (NTS) attenuates ventilatory and sympathetic responses to hemorrhage, and peripheral chemoreceptor stimulation. Thus, we propose that serotonin is released in the NTS during hemorrhage to activate 5-HT1A receptors which facilitate chemoreflex responses. We utilized viral mediated knockdown of the rat 5-HT1A receptor in the caudal NTS (cNTS) to examine the role of 5-HT1A receptors on compensation following hemorrhage and ventilatory responses to hypoxic/hypercapnia. Genes coding a 5-HT1A shRNA or scrambled version of the same sequence were incorporated into adeno-associated viral vectors and injected into the cNTS of rats. After 4 weeks, 5-HT1A shRNA injected rats showed reduced cumulative minute ventilation during hypoxic hypercapnia compared to controls (7,169 ± 366 vs 9,081 ± 754 ml/100g, P(Back to top)
Direct Conversion of Adult Spinal Cord-Derived Oligodendrocyte Progenitor Cells to Functional Neurons
Bazarek S, Marr RA, Briggs CA, Stutzmann GE, Howard RM, Whittemore SR, Peterson DA. (Center for Stem Cell and Regenerative Medicine, Department of Neuroscience, the Chicago Medical School at Rosalind Franklin University of Medicine and Science, IL)
The lack of cell replacement following neurological injury limits the regenerative response of the CNS. Progress in understanding the biology of neural stem cells has raised interest in using stem cells for replacing neurons lost to injury or to disease. As existing committed and uncommitted cells in the CNS do not naturally progress to a neuronal fate, it will be necessary to engineer a conversion to a neuronal fate. Advances in cellular reprogramming provide new tools for re-specification of cell fate and provide a potential alternative to cell transplantation, namely the direct in vivo conversion of resident CNS cell populations for neuronal replacement. Success in this approach will require the generation of relevant neuronal subtypes. The aim of this study was to evaluate the effect of various neurogenic transcription factors (sox2, mash1, olig2VP16, pax6, and neurogenin2) that are related to cell specification during development on fate induction and subtype specificity on resident glia in the spinal cord. An O4+ Oligodendrocyte Progenitor Cell (OPC) population was enriched from the adult rat spinal cord using Magnetic Activated Cell Sorting (MACS). OPCs are the most abundant cycling population in the adult CNS and their isolation provides an ideal in vitro assay for screening neuronal fate determinants. Retroviral-GFP delivery of the single factor, neurogenin2, or the combination of sox2 and mash1 to cultured OPCs resulted in co-expression of GFP with the early neuronal marker, B-III-tubulin at 3 days. Neurogenin2-transduced cells expressed mature neuronal markers, NeuN and MAP2 at 7 days. Repetitive action potentials were evoked by 2 weeks post-transduction. In showing that adult-derived OPCs can be directly converted to functional neurons in vitro, we provide support for investigating the direct in vivo conversion of resident OPCs to a neuronal fate. This approach may provide an alternative therapeutic strategy for neuronal replacement in the adult CNS. (Back to top)
Repeated adolescent CB1 receptor activation impairs the maturation of prefrontal cortical GABAergic function in adulthood
Daryn K. Cass, Eden Flores-Barrera, Daniel R. Thomases, Webster F. Vital, Adriana Caballero, Kuei Y. Tseng (Department of Cellular and Molecular Pharmacology, the Chicago Medical School at Rosalind Franklin University of Medicine and Science, IL)
Converging epidemiological studies indicate that cannabis abuse during adolescence increases the risk of developing psychosis and prefrontal cortex (PFC)-dependent cognitive impairments later in life. However, the mechanisms underlying the adolescent susceptibility to chronic cannabis exposure are poorly understood. Given that the psychoactive constituent of cannabis binds to the CB1 cannabinoid receptor, the present study was designed to determine the impact of a CB1 receptor agonist (WIN) during specific windows of adolescence on the functional maturation of the rat PFC. By means of local field potential recordings and ventral hippocampal stimulation in vivo, we found that a history of WIN exposure during early (postnatal days – P35–40) or mid-(P40–45) adolescence, but not in late adolescence (P50–55) or adulthood (P75–80), is sufficient to yield a state of frequency-dependent prefrontal disinhibition in adulthood comparable to that seen in the juvenile PFC. Remarkably, this prefrontal disinhibition could be normalized following a single acute local infusion of the GABA-Aα1 positive allosteric modulator Indiplon, suggesting that adolescent exposure to WIN causes a functional downregulation of GABAergic transmission in the PFC. Accordingly, in vitro recordings from adult rats exposed to WIN during adolescence demonstrate that local prefrontal GABAergic transmission onto layer V pyramidal neurons is markedly reduced to the level seen in the P30–35 PFC. Together, these results indicate that early and midadolescence constitute a critical period during which repeated CB1 receptor stimulation is sufficient to elicit an enduring state of PFC network disinhibition resulting from a developmental impairment of local prefrontal GABAergic transmission. (Back to top)
Local prefrontal shRNA knockdown of parvalbumin expression is sufficient to reproduce the deficits in prefrontal cortical inhibition elicited by early adolescent treatment with MK-801
Thomases, DR, Flores-Barrera, E, Cass, DK, Caballero, A, Tseng, KY. (Department of Cellular & Molecular Pharmacology, RFUMS/The Chicago Medical School, IL)
A developmental disruption of prefrontal cortical (PFC) inhibitory circuits is thought to contribute to the adolescent onset of cognitive deficits observed in schizophrenia. However, the mechanisms underlying such changes remain unclear. At the cellular level, changes in PFC function are dependent on local dopamine-glutamate interactions and the activity of parvalbumin-positive (PV+) fast-spiking GABAergic interneurons, which exert feed forward inhibition over pyramidal output cells and enable synchronous firing in the PFC. Given the crucial role of N-methyl D-aspartate (NMDA) receptors in the regulation of GABAergic transmission in the PFC, we examined how repeated administration of the NMDA antagonist MK-801 during early adolescence (postnatal days -P35-40-) impacts the normal development of local prefrontal GABAergic function. Electrophysiological analyses of PFC network activity in vivo indicated that early adolescent MK-801 exposure results in an enduring disinhibition of the prefrontal local field potential response to ventral hippocampal stimulation at 20 Hz and 40 Hz when measured in adulthood (P65-85). We found that acute local administration of the GABA-Aα1 positive allosteric modulator Indiplon into the PFC normalized the abnormal prefrontal disinhibitory state induced by early adolescent MK-801 exposure. In vitro whole cell patch-clamp recordings revealed that our in vivo observations are accompanied by a decrease in GABAergic transmission onto layer V pyramidal output neurons. In order to determine if a loss of PV+ GABAergic interneuron function may be responsible for these deficits, we examined the effects of a local prefrontal shRNA-induced knockdown of PV expression in late adolescence (P50). Notably, the immature PFC state produced by early adolescent MK-801 is mimicked by the shRNA knockdown of PV expression resulting in attenuated suppression of the prefrontal local field potential response in vivo and reduced prefrontal GABAergic transmission in vitro. Together these results indicate a critical role of NMDA receptors in regulating the early adolescent maturation of prefrontal GABAergic circuits and point towards a mechanism of impaired PV+ GABAergic interneuron function in the adult PFC following early adolescent exposure to MK-801. (Back to top)
Classical and Alternative Activation of Cyanobacterium Anabaena sp. Lipopolysaccharide (LPS)- treated Rat Brain Microglia
David W. MacAdam, Christopher Osterbauer, Mary L. Hall, Domonkos Feher, Philip Williams, and Alejandro M.S. Mayer (Department of Pharmacology, CCOM, Midwestern University, IL and Department of Chemistry and Biochemistry, University of Hawaii at Manoa, HI)
Cosmopolitan Gram-negative cyanobacteria may contaminate freshwater by releasing toxins, such as lipopolysaccharide (LPS), thus affecting human health. We reported that cyanobacterium Anabaena sp. lipopolysaccharide (AnaLPS) elicited classical activation of rat microglia (BMG) and chemokine MIP-1α and cytokine IL-6 release in vitro (The Toxicologist CD 132 (S-1), 2013). We hypothesized that AnaLPS would activate classical and also alternative activation of BMG in vitro and concomitant release of cytokines and chemokines. AnaLPS was prepared by hot phenol/water extraction. E. coli LPS (EcLPS) 026:B6 from Difco Lab, Detroit, MI was used as a positive control. BMG were isolated from neonatal rats, and treated in vitro with either AnaLPS or EcLPS in a concentration-dependent manner for 18 hours at 35.9 °C. Cytokines and chemokines were determined using a Milliplex® MAP rat cytokine/chemokine multiplex immunoassay. Results were the following: EcLPS and AnaLPS stimulated statistically significant and concentration-dependent release of (a) Pro-inflammatory cytokines: IL-6>IL-1>TNF-; (b) Pro-inflammatory chemokines: MIP-2(CXCL2)>CINC(CXCL1)>MIP-1(CCL3)>MCP-1(CXCL2)>IP-10(CXCL10)>RANTES(CCL5); and (c) Anti-inflammatory cytokine: IL-10, at > than 1ng/mL and 104ng/mL, respectively. We conclude that after an 18 hour in vitro stimulation AnaLPS stimulated both classical and alternative activation of rat brain microglia, but was considerably less potent than EcLPS. Continued investigation of the mechanism responsible for the differential response observed with AnaLPS on BMG is currently ongoing in our laboratory. Support by Midwestern University and the University of Hawaii at Manoa is gratefully acknowledged. (Back to top)
The potential role of endothelial Kir channels in dyslipidemia-induced endothelial dysfunction
Ahn SJ, Bian JT, Han H, Schwabb S, Rosenhouse-Dantsker A, Thomas J, Eddington D, Phillips S, and I. Levitan (Department of Bioengineering, University of Illinois at Chicago, Department of Physical Therapy, University of Illinois at Chicago, Department of Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago)
Dyslipidemia-induced endothelial dysfunction has a major role in the early stage of the development of atherosclerosis. We have shown in our previous studies that increase of cellular cholesterol level subdues the function of endothelial inwardly-rectifying K+ channels (Kir), a major type of endothelial ion channels and a putative flow sensor. In this study, we tested that elevated level of LDL, which is a major cholesterol carrier in the blood, suppressed the endothelial Kir in human aortic endothelial cells. We observed that the endothelial Kir was dose-dependently suppressed by the increased LDL level; specifically, from <100 to 150 mg/dl, the Kir activity was sharply dropped. We also showed that this suppression could be reversed by HDL (50 mg/dl). Therefore, we propose that the activity of Kir is vigorously coordinated by altering the LDL/HDL ratios from normal to pathological levels. Additionally, we tested the function of Kir suppression on agoist- and flow- induced vasodilatation. Three complementary strategies are used to achieve this goal: (i) use of lentiviral construct of a dominant-negative Kir2.1 subunit that subdues Kir activity, (ii) comparing mesenteric arteries isolated from Kir2.1+/- and WT mice and (iii) applying the Kir channel blocker (Ba2+) to the arteries. Our observation showed that the reduced Kir activity significantly interfered acetylcholine- and flow- induced vasodilatation in murine mesenteric arteries.
* The zip code of all departments in University of Illinois at Chicago is as same as 60612.
** All works were performed in University of Illinois at Chicago. (Back to top)
Disturbed Shear Stress Increases Caveolae-Mediated oxLDL Uptake Leading to Endothelial Cell Stiffness
Elizabeth LeMaster, Shan Sun, David Eddington, Michael Cho, Rich Minshall and Irena Levitan (University of Illinois at Chicago, Departments of Bioengineering, Pharmacology, Anesthesiology, and Pulmonary, Critical Care and Sleep Medicine)
Atherosclerosis is a major risk factor in the development of cardiovascular disease, which is the leading cause of death in the United States. Endothelial cell (EC) dysfunction is one of the initiating steps in the development of atherosclerotic plaques, which preferentially form at the disturbed shear stress (DSS) regions of vascular bifurcations. Earlier studies show that oxidized low density lipoprotein (oxLDL), an established risk factor for plaque formation, induces an increase in EC stiffness in vitro.
This study tested the impact of different hemodynamic conditions on endothelial cell uptake of oxLDL. A flow channel with a single step occlusion was designed, tested and microfabricated to create both unidirectional laminar and recirculating disturbed shear stress patterns. My data show that oxLDL uptake into ECs increases in DSS regions and is significantly reduced by knocking out (KO) caveolin-1 (Cav-1), the main protein constituent of caveolae. This indicates that endocytosis via caveolae, or flask-like membrane invaginations, play a major role in oxLDL uptake into ECs and is consistent with studies showing that Cav-1 KO mice do not develop atherosclerotic plaques.
Furthermore, my studies show that this increased oxLDL uptake in DSS regions promotes endothelial stiffness, as assessed by atomic force microscopy. This data is further supported by ex vivo stiffness measurements from intact mouse vessels. There is increased EC stiffness in the DSS regions of the aortic arch as compared to the laminar shear stress regions of the descending aorta. (Back to top)
Oncolytic Adenovirus as a potential tool for pancreatic cancer combination therapy
Amanda Oliveira, Christopher LaRocca, Joohee Han, Ryan Shanley, Julia Davydova, and Masato Yamamoto (Department of Surgery, University of Minnesota, Minneapolis)
Current effective therapies against pancreatic cancer are limited, resulting in patient five-year survival rate of only 6%. Combination therapies have been shown to be promising in the treatment of pancreatic cancer: adjuvant therapies combining chemoradiation and systemic IFN alpha (IFN) were reported to increase five year survival of patients to 40 -55%. Despite impressive survival results, low half-life of systemically administered IFN inhibited achieving sufficient concentrations of the cytokine at the tumor site. Considering IFN is a chemoradio sensitizer, low IFN intra-tumor levels might have hampered the therapeutic effect of combination therapy, highlighting the need for a better IFN delivery method.
We hypothesize that Adenovirus (Ad) can improve this therapy via high and local intra-tumor expression of an effector gene. We constructed an oncolytic adenovirus (OAd) vector expressing human IFN from the adenoviral E3 region (OAd-IFN-hum). To enhance adenoviral potency and infectivity, we overexpressed adenoviral death protein (ADP) and included chimeric Ad5/Ad3 fiber, respectively. We analyzed in vivo vector effectiveness in immunocompetent syngeneic hamster pancreatic cancer model with a vector encoding hamster IFN (OAd-IFN-ham) and RGD fiber modification.
In vitro studies testing functionality of the OAd-IFN vectors in human and hamster pancreatic cancer demonstrated that overexpression of ADP remarkably improved oncolysis. OAd-IFN vectors significantly increased cytocidal effect when compared with an identical counterpart expressing luciferase (OAd-Luc). The IFN-expressing viruses sensitized the cells to 5-FU, Cisplatin, and Gemcitabine, and increased their susceptibility to radiation (4 and 8 Gy).
In hamster pancreatic cancer subcutaneous model, the groups treated with OAd-IFN+radiation (8 and 20GY), or OAd-IFN+5-FU had superior survival and anti-tumor effects compared to chemoradiation. The triple combination therapy (OAd-IFN+radiation+5-FU) showed most remarkable anti-tumor effect.
The survival and anti-tumor effect data indicated the potential of OAd-IFN for development of more effective pancreatic cancer combination therapy, mediated by local and massive expression of IFN. These data suggest that oncolytic adenovirus not only is a powerful tool to treat pancreatic cancer, but also have high potential to increase effectiveness of therapies where high intra-tumor transgene expression is necessary. (Back to top)
Dysregulated Protein Synthesis Maintain Synaptic Adaptations During Withdrawal from Cocaine Self-Administration
A. F. Scheyer, M. E. Wolf, K. Y. Tseng (Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, IL)
Prolonged withdrawal from extended-access cocaine self-administration is associated with a progressive intensification (“incubation”) of cue-induced cocaine craving that is associated with profound adaptations in medium spiny neurons (MSN) of the nucleus accumbens (NAc). These adaptations include the accumulation of high-conductance, Ca2+-permeable AMPA receptors (CP-AMPAR) and a switch in group I metabotropic glutamate receptor (mGluR) plasticity from primarily mGluR5-mediated transient synaptic depression to mGluR1-mediated long-term depression (LTD). To determine the role of protein synthesis in maintaining these adaptations, we conducted whole-cell patch-clamp recordings in NAc MSN in the presence of the protein translation inhibitors anisomycin, cycloheximide or rapamycin. All recordings were conducted in slices prepared from rats that underwent >45 days of withdrawal from extended-access cocaine self-administration or saline self-administration (controls). At this withdrawal time, the cocaine-exposed group exhibits markedly elevated cocaine craving compared to rats tested immediately after discontinuing cocaine self-administration. To assess the contribution of CP-AMPARs to NAc synaptic transmission, we measured the rectification index and sensitivity to the CP-AMPAR antagonist naspm. Both measures were elevated in slices from cocaine animals recorded under control conditions (aCSF), indicating a significant CP-AMPAR contribution (as expected from our prior studies), whereas incubation of slices with anisomycin or cycloheximide reduced these measures to levels observed in saline control rats. Similarly, protein synthesis inhibition restored mGluR5 function in cocaine-treated rats while abolishing mGluR1-LTD, resulting in a state functionally similar to that of saline controls. Together, our data indicate a critical role for protein synthesis-dependent mechanisms in sustaining the abnormally elevated CP-AMPAR transmission and altered mGluR plasticity found at NAc glutamatergic synapses. As these adaptations are directly linked to persistent enhancement of cocaine craving and resulting vulnerability to relapse, our findings suggest that aberrant regulation of protein translation may be an important contributor to addiction and perhaps a target for pharmacological intervention. (Back to top)
Electrophysiological properties of female basolateral amygdala neurons fluctuate within the estrous cycle and are functionally distinct from males
Shannon R. Blume, Andrea Hetzel and J. Amiel Rosenkranz. (Department of Cellular and Molecular Pharmacology. Rosalind Franklin University of Medicine and Science, North Chicago, IL)
Prolonged periods of psychological trauma and/or chronic stress often precede the onset of affective disorders such as anxiety, depression and post-traumatic stress disorder. Within these disorders, women are at a disadvantage as they are more likely to suffer from an affective disorder in their lifetime compared to men. This difference between females and males may occur as a result of sex differences in limbic function. The basolateral amygdala (BLA) is a limbic structure integral to the generation of emotions, emotional learning and emotion driven behaviors. Despite all that is known regarding sex differences in BLA-dependent behaviors in humans and rodents, very little is known about female amygdala physiology. In this study, BLA neuronal activity was measured using in vivo extracellular single unit recordings in cycling female and male rats. Membrane properties were examined using in vitro whole cell patch clamp recordings in BLA projection neurons. Our in vivo experiments revealed a significant sex difference in BLA neuronal activity. The BLA was more active in females compared to males. BLA neuronal firing properties such as the number of active BLA neurons per electrode track and the coefficient of variation were examined however no sex differences were observed. The relationship between action potential width and BLA neuronal firing rate was more variable in the female (control) BLA compared to males (control), suggesting that females have a more diverse population of spontaneously firing neurons. Immunohistochemistry experiments demonstrate a difference in the number of parvalbumin expressing GABAergic interneurons between males and diestrus and proestrus females. In support of this finding, our in vitro experiments revealed a significant difference in the frequency of spontaneous inhibitory post-synaptic current (IPSC) events between estrous cycle phases and sexes. In addition, lateral amygdala neurons were more excitable in females compared to males. Our findings suggest the BLA in cycling females is functionally distinct from males. The disparity between sexes in BLA neuronal activity and properties may render females more vulnerable to the development of affective disorders. (Back to top)
Colorimetric Tests for Screening Ciprofloxacin on a PAD (Paper Analytical Device)
Elizabeth Robbins, B.S. and Toni Barstis Ph.D. (Saint Mary’s College, IN)
The problem of counterfeit pharmaceuticals is on the rise across the world, and this is especially prevalent in developing countries that lack strong drug regulation and enforcement.1 According to the World Health Organization, 45% of counterfeited drugs were antibiotics between 1982-1999. 2 Many of these antibiotics are sold well past their expiration date and thus do not contain the correct dosage of active ingredient. Ciprofloxacin, a broad-spectrum antibiotic, has been counterfeited by selling substandard quality or expired tablets at full price.1 To address this problem, our research group developed a simple device called the “Cipro PAD” (paper analytical device) to screen suspicious ciprofloxacin tablets. This PAD uses well-established colorimetric tests on a paper-based substrate to identify the active ingredient, ciprofloxacin, as well as its degraded products (e.g., piperazine ring or phenol) found in expired tablets. The reagent ferrous ammonium sulfate is used to test for ciprofloxacin and an iodide-iodine reagent is used to test for starch, a binder used in genuine tablets. Reagents used to test for incorrect binders or fillers and for heavy metals contamination are also included on the Cipro PAD. It is our hope that consumers will be able to use the Cipro PAD to verify the authenticity of their ciprofloxacin tablet.
Bate, Roger, Ginger Zhe Jin, and Aparna Mathur, Counterfeit or Substandard? The Role of Regulation and Distribution Channel in Drug Safety NBER Working Paper No. 18073 May 2012
World Health Organization. Department of Essential Drugs and Other Medicines, Counterfeit Medications: Fact Sheet. http://www.who.int/medicines/services/counterfeit/impact/ImpactF_S/en/index.html. Nov 14, 2006. (Back to top)
Generation of Plasmids Containing DNA-Protein Crosslinks at Specific Loci
Lisa Chesner, Shaofei Ji, Colin Campbell. University of Minnesota-Twin Cities
DNA–protein cross-links (DPCs) form following exposure to a variety of agents, including formaldehyde, ionizing radiation, and several anti-cancer therapeutics, however, little is known about the biological mechanisms of their recognition and repair. The goal of this project is to synthesize defined DPC plasmid substrates to study DNA repair in vitro. Two distinct strategies are being pursued. In the first, a DPC-containing oligonucleotide is annealed to single-stranded pGL3 and a primer extension reaction is performed to generate double stranded molecules. In the second strategy, a duplex DPC-containing oligonucleotide is ligated into a linearized double-stranded plasmid. These covalently closed DPC-containing molecules, purified via CsCl gradients, will be introduced into wild-type and repair deficient human cell lines to perform DNA repair analysis. (Back to top)
RGS6, not RGS4, is the primary Regulator of G protein Signaling (RGS) modulator of the parasympathetic regulation of heart rate
Nicole Wydeven, Ekaterina Posokhova, Zhilian Xia, Kirill Martemyanov, Kevin Wickman (Dept. of Pharmacology, University of Minnesota, MN Dept. of Neuroscience, Scripps Research Institute, Jupiter, FL)
Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K+ channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4-/-, Rgs6-/-, and Rgs4-/-:Rgs6-/- mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6-/- mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6-/- and Gβ5-/- mice, suggest that the partial rescue of phenotypes in Rgs4-/-:Rgs6-/- mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice. (Back to top)
Involvement of CaMKIIα in multiple sclerosis-associated pain
Xiaoyu Hu, Fang Huang, and Zaijie Jim Wang. (Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois at Chicago, IL)
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system, with manifestations of neuroinflammation and demyelination. Pain in MS patients, with remarkable prevalence up to ~90%, significantly reduce quality of life. Over past decades, advances have been made in understanding the neurobiological mechanisms of motor dysfunction in MS, but to a much lesser extent, MS-associated pain. Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα), a multifunctional serine/threonine protein kinase, plays a key role in long-term potentiation and synaptic plasticity. We established a widely used animal model, experimental autoimmune encephalomyelitis (EAE), to study the pain mechanism in mice. We found that KN93, a selective CaMKII inhibitor, effectively prevented and reversed the mechanical and thermal hypersensitivity in EAE mice. Also, acute application of CaMKIIα siRNA significantly attenuated pain severity caused by EAE. Furthermore, CaMKIIαT286A point-mutation mice showed significant resistance to EAE induced hypersalgesia. We also found that the spinal CaMKIIα activity was increased in EAE mice, and reduced by CaMKIIα inhibition, correlating with the behavior results. In sum, our data suggested that CaMKIIα is critically involved in the development and maintenance of pain and neuropathy in multiple sclerosis. Inhibiting CaMKIIα may offer potentially new pharmacological interventions to prevent or attenuate multiple sclerosis-associated pain. (Back to top)
RIP140 contributes to foam cell formation and atherosclerosis by regulating cholesterol homeostasis in macrophages
Yi-Wei Lin*, Pu-Tzu Liu*, Jennifer L. Hall#, Li-Na Wei*, ( *Department of Pharmacology, University of Minnesota Medical School, MN and #Lillehei Heart Institute, University of Minnesota, MN)
Atherosclerosis, a syndrome with abnormal arterial walls, is one of the major causes that lead to the development of various cardiovascular diseases. The key initiator of atherosclerosis is cholesterol accumulation. The uncontrolled cholesterol deposition, mainly involving low-density lipoprotein (LDL), causes atheroma plaque formation, which initiates chronic inflammation due to the recruitment of inflammatory cells such as macrophages. Macrophages scavenge excess peripheral cholesterol and transport intracellular cholesterol to high-density lipoprotein (HDL) for excretion or storage. Cholesterol-laden macrophage-derived foam cell formation is the main cause of atherogenesis. It is critical to understand the regulatory mechanism of cholesterol homeostasis in the macrophage in order to prevent foam cells formation and further develop novel therapeutic strategies against atherosclerosis. Here we identified a protein, RIP140 (receptor interacting protein 140), that promotes cholesterol accumulation in the macrophage by reducing reverse cholesterol transport within macrophages, resulting in foam cell formation. In animal models, we found that reducing RIP140 levels, by either bone marrow transplantation from macrophage-specific RIP140 knockdown (RIP140-M-KD) mice to ApoE null mice, or crossing RIP140-M-KD with ApoE null mice, effectively ameliorates high-cholesterol diet-induced atherosclerosis. This study provides a proof-of-concept for RIP140 as a risk biomarker of, and a therapeutic target for, atherosclerosis. (Back to top)
Non-dopaminergic Mechanisms Underlying the Frequency-dependent Corticostriatal Disinhibition Following Chronic Dopamine Lesion in a Rodent Model of Parkinsonism
Vatsala R. Jayasinghe, Anthony R. West, Kuei Y.Tseng (Department of Cellular and Molecular Pharmacology, Department of Neuroscience, RFUMS/The Chicago Medical School, IL)
Parkinson’s disease is strongly associated with the emergence of increased beta oscillations within the basal ganglia, which is thought to reflect a disruption of corticostriatal transmission, and thereby exacerbate motor deficits associated with this disease. The mechanisms underlying the elevated synchronization at beta frequencies observed following dopamine lesion remain unclear. However, dysfunction of local GABAergic interneurons may play a key role as these cells provide robust feed-forward inhibition to striatal projection neurons, and this inhibition is impaired in the dopamine-depleted, parkinsonian striatum. Furthermore, elevated cGMP signaling, which facilitates cortico-striatal transmission, is indicated in the parkinsonian striatum. Therefore, the goal of this study was to determine if impaired GABAergic function or elevated cGMP signaling could underlie the increased synchrony within the corticostriatal pathway at specific frequencies observed in the parkinsonian state. In vivo electrophysiological recordings performed in the dorsal striatum of rats revealed a progressive increase in the inhibitory modulation of the local field response evoked via motor cortex train stimulation at 10 (alpha), 20 (beta), and 40 Hz (gamma). Interestingly, this frequency-dependent striatal inhibition evoked during cortical stimulation was lacking in the dopamine-depleted striatum of rats that received the unilateral 6-OHDA lesion. We next asked whether local GABAergic transmission controls striatal responsivity to cortical drive in a frequency dependent manner. Local infusion of the GABA-AR antagonist picrotoxin into the dopamine-intact striatum revealed a frequency-dependent disinhibition of corticostriatal transmission which was similar to that seen in the dopamine-depleted striatum. A similar effect was observed with the introduction of elevated cGMP levels in the striatum. Together, these results highlight the possibility of an impaired striatal GABAergic inhibitory control and/or cGMP signaling in the dopamine-depleted striatum that enables the augmented cortico-basal ganglia synchronization that spreads beyond the beta oscillation range. (Back to top)
Generating new drug leads for schistosomiasis from regenerating flatworms
John D. Chan & Jonathan S. Marchant. (Department of Pharmacology, University of Minnesota; Minneapolis, MN)
Schistosomiasis is a disease caused by a parasitic flatworm infection. There are an estimated ~200 million people infected worldwide, a burden that encumbers third world economies with an annual loss of several million disability-adjusted life years. The drug praziquantel (PZQ) is the key clinical therapy but the biological target of this drug is unclear. To bring new insight to this problem, we have been investigating the mechanism of action of PZQ during regeneration of free-living planarian flatworms, where the drug effects an axis duplication to yield two-headed animals. In this system, we evidence PZQ activation of a neuronal Ca2+ channel modulates opposing dopaminergic and serotonergic pathways to regulate ‘head’ structure formation. Remarkably, compounds with efficacy for either bioaminergic network in planarians also displayed antischistosomal activity, and reciprocally, agents first identified as antischistocidal compounds caused bipolar regeneration in the planarian bioassay. We propose the distinct phenotypes - PZQ-evoked bipolarity in planarians and PZQ-evoked toxicity against schistosomes - represent unexpected yet orthologous phenotypes (‘phenologs’) resulting from engagement of the same fundamental Ca2+-triggered bioaminergic interactome in each system. This discovery underscores the utility of basic research on axis patterning mechanisms in the tractable planarian system for the discovery of novel antischistosomal drug leads, and more broadly mechanistic insight into the signaling pathways engaged by PZQ, a key human therapeutic. (Back to top)
Protein Kinase Cα is a novel regulator of FOXC2 in the basal A triple negative breast cancer
Thao ND Pham, Bethany P. White, Debra A. Tonetti. (Department of Biopharmaceutical, UIC, Chicago, IL)
Breast cancer is the second leading cause of cancer death in women. Accounted for about 20% of all breast cancer cases, triple negative breast cancer (TNBC) remains a clinical challenge due to the lack of targeted therapies. The identification of pathways specific to TNBC will be tremendously helpful in generating new molecular targets and improving patient outcomes. Our lab has shown that Protein Kinase Cα (PKCα) expression is linked to endocrine resistance (Tonetti et. al. 2001), and the TNBC subtype of clinical samples (Tonetti et. al. 2012). We demonstrated that PKCα mediates a novel epithelial-mesenchymal-transition (EMT), which is generally considered the initiation of metastasis for cancer progression, in T47D/PKCα cells through a mechanism that involves upregulation of FOXC2 and repression of p120-catenin (White et. al., AACR 2011) instead of E-cadherin as seen in the canonical pathway of EMT. Recently, we found that both PKCα and FOXC2 are highly expressed in the basal A subgroup of TNBC (HCC1937 and HCC1143) both at the protein and mRNA levels. We hypothesized that PKCα may govern the aforementioned novel EMT in these cell lines. Indeed, upon treatment with TPA, a PKC activator , we observed a significant increase in the mRNA levels of Foxc2 in these cells. Moreover, PKCα knockdown by siRNA reduced transcripts and protein expression of Foxc2. Here, we report for the first time that PKCα regulates FOXC2 in the TNBC basal A subgroup, and may therefore regulate a novel EMT in these cell lines. (Back to top)
The ESCRT machinery mediates Akt signaling induced by the G protein-coupled receptor CXCR4
Rita Verma and Adriano Marchese (Program in Molecular Biology, Department of Pharmacology and Experimental Therapeutics, Stritch School of Medicine, Loyola University Chicago, IL)
The G protein-coupled receptor CXCR4 and its cognate ligand CXCL12 have been shown to protect against myocardial ischemia and reperfusion injury via Akt, a protein kinase linked to cell survival. However, the molecular mechanisms that govern CXCR4-induced Akt signaling remain poorly understood. The objective of this study is to elucidate the molecular mechanisms that mediate CXCR4- induced Akt activation. Activated CXCR4 is rapidly ubiquitinated and internalized from the plasma membrane onto endosomes where it is sorted for lysosomal degradation via the ESCRT (Endosomal Sorting Complex Required for Transport) machinery. While this leads to downregulation of receptor signaling, we show here that the ESCRT machinery also has a role in positively regulating CXCR4 signaling. We show that CXCR4-induced phosphorylation of Akt on serine 473 (Ser-473) is attenuated in HeLa cells treated with siRNA directed against subunits of ESCRT-0, -I, -II and –III complex. Accordingly, this leads to a decrease in Akt signaling as monitored by immunoblotting to detect phosphorylation of Foxo 1/3a, an Akt phosphorylation site. Fixed cell confocal fluorescence microscopy analysis reveals that myc-Akt colocalizes with ESCRTs on EEA-1-positive endosomes. Similarly, Rictor, an important component of the mTORC2 complex, the kinase that phosphorylates Akt-S473, is also found on EEA-1- positive endosomes containing ESCRTs. These data suggest that ESCRTs facilitate the phosphorylation of Akt-S473 by mTORC2 on endosomes upon CXCR4 activation. (Back to top)
BTB-ZF TRANSCRIPTION FACTORS RECRUIT THE E3 UBIQUITIN LIGASE CULLIN 3 TO INITIATE LYMPHOID EFFECTOR PROGRAMS
Rebecca Mathew, Michael P. Seiler, Seth T. Scanlon, Michael G. Constantinides, Clara Bertozzi-Villa, Jeffrey D. Singer and Albert Bendelac (Committee on Immunology, The Howard Hughes Medical Institute, University of Chicago, IL, Department of Biology, Portland State University, OR)
The differentiation of several T and B cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodeling. We report that PLZF, the BTB-ZF transcription factor directing the innate-like effector program of NKT thymocytes was prominently associated with cullin 3 (Cul3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF induced the transport of Cul3 from the cytosol to the nucleus where the two proteins were associated within a chromatin associated/modifier protein complex. Cul3 was also found associated with another BTB-ZF transcription factor, Bcl6, which directs the B cell germinal center program. Conditional deletion in mice demonstrated an absolute, cell-intrinsic requirement of Cul3 for the development of NKT cells and germinal center B cells. We conclude that distinct lineage-specific BTB-ZF transcription factors recruit Cul3 to alter the ubiquitination pattern of their associated chromatin associated/modifier complex and thereby directing the differentiation of several T and B lymphocyte effector programs. We propose that this novel function may also be involved in the oncogenic role of PLZF and Bcl6 in leukaemias and lymphomas. (Back to top)
Developmental regulation of ventral hippocampal-basolateral amygdala interactions in the prefrontal cortex
Meyer, JD, Thomases, DR, & Tseng, KY (Department of Cellular & Molecular Pharmacology, RFUMS/The Chicago Medical School, North Chicago, IL , Lake Forest College, IL)
The functional maturation of the prefrontal cortex (PFC) is dependent upon proper integration of glutamatergic inputs from the ventral hippocampus (vHipp) and the basolateral amygdala (BLA). We have recently determined that inputs arising from the ventral hippocampus and the BLA elicit distinct forms of long-term plasticity in the PFC in an age-dependent manner. In particular, ventral hippocampal stimulation produces a GABA-dependent until late adolescence. In contrast, a phenotype of long-term potentiation (LTP) elicited from the BLA is already present in the adolescent PFC and does not require local GABAergic transmission. Given the critically interconnected roles of the vHipp and the BLA in mediating PFC functions we asked the question whether the maturation of hippocampal-dependent plasticity in the PFC coincides with the acquisition of augmented inhibitory regulation of the prefrontal response to BLA inputs by the vHipp. Using in vivo local field potential recordings we determined that following the normal induction of BLA-evoked prefrontal LTP in adult rats (post natal day -P- 60-80) subsequent high frequency stimulation of the ventral hippocampus resets the BLA LTP to baseline. However, such heterosynaptic suppression is lacking in early adolescent (P30-40) rats. Similarly, high frequency stimulation of the vHipp preceding that of the BLA prevents subsequent BLA-induced LTP in adult rats, an interaction that is not yet present during early adolescence. Administration of the NMDA receptor antagonist MK-801 during early adolescence prevents the normal development of vHipp-dependent PFC plasticity. Therefore, we examined the effects of early adolescent MK-801 on the maturation of vHipp-dependent inhibition of BLA-evoked prefrontal plasticity. When tested in adulthood, following early adolescent exposure to MK-801, adult rats exhibit the phenotype of a functionally juvenile PFC without the normal inhibition of BLA-dependent prefrontal LTP. Together these results indicate that the maturation of the vHipp-PFC pathway coincides with the acquisition of vHipp- dependent inhibitory regulation of BLA-induced prefrontal plasticity. Furthermore this maturation is activity-dependent and can be prevented by the transient blockade of NMDA receptors by MK-801 during early adolescence. (Back to top)
G protein-coupled receptor kinase selective small molecule inhibitors
Kelly Larimore, Kristoff T. Homan, Helen Waldschmidt, Emily Wu, Michael Wilson, David M. Thal, Scott D. Larsen , and John J. G. Tesmer (Departments of Biological Chemistry and Pharmacology, Life Sciences Institute, Vahlteich Medicinal Chemistry Core and the Department of Pharmacology, University of Michigan) 48109
G protein-coupled receptor kinase 2 (GRK2) and GRK5 inhibition have become potential therapeutic routes for the treatment of heart failure and cardiac hypertrophy. Though there are no commercially available small molecule therapeutics selective for either of these kinases, high-throughput screening using GRKs 1, 2, and 5 have identified first generation small molecule inhibitors with low-micromolar potency and substantial selectivity between GRKs. The most potent of these compounds have also been shown to increase contractility in whole animal and isolated cardiomyocytes, therefore showing that these small molecules hold potential in becoming viable therapeutics. Using X-ray crystallography, we have solved several GRK-scaffold complexes that reveal mechanisms of selectivity among GRKs and AGC kinases. Based on these structures, repeated rounds of rational design have given rise to compounds with nanomolar potency and high subfamily selectivity, providing greater understanding into the structure activity relationships. These compounds form the basis of a novel ‘GRK inhibitor toolkit’ that can be used to decode GRK roles in cellular signaling and influence the development of novel therapeutics. (Back to top)
Uncovering the Contributions of Amino Domain and Alternatively Spliced Variants of Alpha-Synuclein in Parkinson’s Disease: Insights from Yeast
K. Hamid*, S. Bello Rojas*, L. Graham*, C. Gudmundson*, A. McCracken*, P. Schrag*, N. Kukulka, and S. DebBurman (*Undergraduate researchers Biology Department, Neuroscience Program, Lake Forest College, IL)
Parkinson's disease (PD) is a hypokinetic neurodegenerative disorder characterized by the death of midbrain dopaminergic neurons. This cell death is linked to the accumulation of an aggregated protein, a-synuclein, which is 140 amino acids long (Syn-140). This protein has three functional parts, one of them being the N-terminal domain (amino acids 1-60), which is associated with lipid binding. The amino acids within this domain that are critical for lipid binding are not clear. Moreover, three shorter alternatively spliced forms of a-synuclein (Syn-126, Syn-112, and Syn-98) that are missing parts of the N-terminal and C-terminal domains were recently discovered in normal and PD patients; however their contributions to health and disease need further evaluation. This present study consists of two projects, both being evaluated in a budding yeast model for PD developed by the DebBurman lab: 1) characterization of the splice variant forms of a-synuclein, and 2) evaluation of N-terminal domain by creation of six truncation mutants. We have synthesized the three splice variants for yeast expression and are testing the hypothesis that they will contribute to PD, by decreasing membrane association (Syn-126, Syn-98) and solubility (Syn-112, Syn-98). We have also created six N-terminal truncation mutants (Syn 10-140, Syn 20-140, Syn 30-140, Syn 40-140, Syn 50-140 and Syn 60-140) and are testing the hypothesis that specific smaller regions within the N-terminal domain govern lipid binding, protein aggregation, and toxicity of a-synuclein. Better understanding of the amino domain and the splice variants of a-synuclein will contribute to the overall understanding of PD pathogenesis. (Back to top)
Unexpected Insights from Yeast into C-terminal Truncations of -Synuclein: Relevance to Parkinson’s Disease
C. Alvarado, K. Campbell, S. Chiren, J. James, M. Munoz, A. Roman, and S. DebBurman. (Biology Department and Neuroscience Program, Lake Forest College, IL)
Parkinson's disease (PD) is a neurodegenerative disorder characterized by proteinaceous aggregates called Lewy bodies in dying dopaminergic neurons in the midbrain; the aggregated protein is -synuclein (-Syn), a 140-amino acid abundant neuronal protein that regulates vesicular traficking. In PD patients and healthy individuals, -Syn is found truncated at the C-terminus. Investigations in vitro and in mammalian cell culture demonstrate that these truncation variants are more aggregation prone and toxic to cells. Confirmation of these properties in organismal PD models is lacking, as is the influence of truncations on the several familial mutants forms of -Syn. Using our well-established budding yeast model for -syn expression, we created and compared four C-terminally truncated versions (Syn-123, Syn-120, Syn-113, Syn-110) with the full-length Syn-140. We made this comparison with wild-type and three familial mutants of the protein (specifically, A30P, E46K, and A53T. All variants were tagged with green fluorescent protein (GFP) at the C-terminus, but the wild-type and A53T versions were also tagged with GFP at the N-terminus to confirm that our characterization of -Syn localization did not depend on the location of GFP tag. We have begun analyzing a total thirty -Syn variants for two properties: localization (using GFP microscopy) and toxicity (using yeast spotting and survival assays) to test the hypothesis that larger truncations will lead to increased protein aggregation and toxicity because -Syn's C-terminus governs solibility. We have already gained several unexpected insights through our initial microscopy data: C-terminal truncations regulate 1) -Syn membrane binding (a property linked with -Syn's amino domain); 2) -Syn forms more small cytoplasmic foci and is excluded from vacuoles; 3) The location of GFP tag does influence the extent to which the truncations affect both these properties. We are currently conducting our toxicity analysis, and in the future with student aggregation and accumulation of -Syn through Western blotting and protein turnover assays. (Back to top)
Engineering pH-Dependent Anti-Hapten Antibodies: Exploring Function/Stability
Willie E. Carter, Christopher Smith, (Loyola University Chicago, IL)
The Horn research group is interested in the developing novel protein affinity reagents. Antibodies, one of the most prevalent affinity reagents, show great potential for therapeutic and diagnostic use. Our engineering efforts aim to modulate the binding and stability of a unique heavy-chain only antibody, called VHH. To generate a pH-dependent antibody interaction, a histidine substitution was introduced within the anti-methotrexate interface. A consequence of the newly engineered pH-dependent VHH antibody was a modest decrease in methotrexate binding affinity at the permissive pH, as well as a loss in conformational stability. Here we explore whether an engineered disulfide bond within the VHH scaffolding may help counter these energetic penalties. Our findings reveal that the relationship between engineering new function and loss of old function/stability is likely to be a common phenomenon. (Back to top)
Molecular Mechanisms of Phospholipase C-β Autoinhibition
Jessica A. Begley, Angeline M. Lyon, Taylor Manett, and John J. G. Tesmer. (Life Sciences Institute and the Departments of Pharmacology and Biological Chemistry, University of Michigan, Ann Arbor, MI)
Phospholipase C β (PLC β) hydrolyzes the inner membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), generating the second messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG), which are key regulators of intracellular Ca2+ levels and protein kinase C (PKC) activity. These downstream effects of PLCβ have profound consequences for muscle contraction in the cardiovascular system, and therefore, the PLC β enzymes are tightly regulated. Under basal conditions, PLC β enzymes have very low basal activity, which is achieved by autoinhibition mediated by a flexible loop in the catalytic core (the X–Y linker) and by a highly conserved autoinhibitory helix in the C-terminal regulatory region of PLC β. Mutations in these regions results in increased basal activity and decreased thermal stability. The X-Y linker is thought to contribute to low activity by occluding the substrate from the active site. The C-terminal helix docks in close proximity to the linker and the active site, however, the molecular basis for how it regulates PLC activity remains unclear. We have used mass spectrometry, site-directed mutagenesis, and biochemical assays to begin to elucidate the mechanism of autoinhibition of lipid hydrolysis by these various regulatory elements. By identifying the molecular basis of PLC β autoinhibition, a more comprehensive understanding of PLCregulation and activation will be obtained, providing new insights into the design of novel therapies to treat heart failure.
Support for this work was provided by NIH grants HL086865 and HL071818 (J.J.G.T.) and an American Heart Association Post-Doctoral Fellowship 13POST1637009 (A.M.L.). (Back to top)