The Cardiomyocyte Circadian Clock Directly Regulates b-Hydroxybutarate Dehydrogenase Activity

SIBS student – B. J. Ammons

SIBS mentor – Timothy Kraft

Introduction: Circadian clocks are cell autonomous molecular mechanisms that confer the selective advantage of anticipation. Critical to circadian clock function are two transcription factors, CLOCK and BMAL1. Through use of two distinct genetic mouse models of cardiomyocyte clock disruption (cardiomyocyte-specific CLOCK mutant [CCM] and cardiomyocyte-specific BMAL1 knockout [CBK] mice) we have begun to highlight novel roles for this mechanism in both myocardial physiology and pathophysiology.  Recent gene expression microarray and proteomic studies using CCM and CBK models identified b-hydroxybutarate dehydrogenase 1 (BDH1) as being cardiomyocyte circadian clock regulated.  Whether changes in BDH1 gene and protein expression translate to functional changes in b-hydroxybutarate dehydrogenase activity is currently unknown.

Hypothesis: The cardiomyocyte circadian clock regulates myocardial ketone body metabolism through direct regulation of b-hydroxybutarate dehydrogenase activity.

Methods/Results: Spectrophotometric assays for both b-hydroxybutarate dehydrogenase and citrate synthase (control) were initially established and validated (e.g., substrate dependence).  Hearts were isolated from adult CCM and CBK, as well as littermate wild-type, mice.  b-Hydroxybutarate dehydrogenase activity was found to be markedly lower in both CCM and CBK hearts, relative to their respective littermate wild-type hearts.  In contrast, citrate synthase activity was not significantly different in either CCM or CBK relative to their wild-type controls.

Conclusions: The cardiomyocyte circadian clock directly regulates cardiac CCM and CBK b-hydroxybutarate dehydrogenase activity. These data are consistent with the concept that the cardiomyocyte circadian clock confers the selective advantage of anticipation of prolonged fasting, when the animal in the wild is unsuccessful in its forage for food during the active period.

The Role of Langerhans Cells in Carcinogenesis of the Skin

SIBS student - Meg Apperson

SIBS Mentor - Dr. Laura Timares

Langerhans cells are dendritic cells located in the epidermis of the skin, where they serve as skin-specific antigen presenting cells after migrating to skin-draining lymph nodes. The specific role of these cells has been further investigated after the development of murine models designed to ablate Langerhans. Transgenic mice have been developed in which diphtheria toxin (DT) is expressed under control of the human langerin gene promoter, affecting the subset of langerin expressing cells in the epidermis. This murine model has allowed for better understanding of the Langerhans cells’ role in immunity, including that they seemingly have the ability to both activate and suppress immune responses. An area of interest is how these Langerhans cells participate in UV and chemical-induced carcinogenesis of the skin, possibly by regulating immune responses.  To validate the mouse phenotypes, with respect to Langerhan cells in the skin, we mated Langerin DTA expressing heterozygous male with wild-type (C5713L6) females and the progeny was genotyped for the Langerin DTA gene. Mice that did not express the gene served as wild-type controls. We found that the mice that expressed langerin DTA gene lacked Langerhan cells, as indicated by MHC Class II stains of the epidermis. Langerin DTA mice showed exaggerated ear swelling response against dinitrofluorobenzene as compared to WT mice. Since IL-12 and IL-23 are two important cytokines in DC activity, we indicated there was a difference in Langerhan cell density with cytokine ablation. The findings showed there was a lower Langerhan cell density when both cytokines were absent.

Identifying the pathway by which glucose induces cardiomyocyte TXNIP expression

SIBS student - Rebecca Duron

SIBS Mentors - Junqin Chen and Anath Shalev

Thioredoxin-Interacting Protein (TXNIP) has been shown to have harmful effects on the cardiovascular system by inducing oxidative stress, cardiomyocyte inflammation, and subsequent cardiomyocyte apoptosis. Because TXNIP is upregulated under conditions of high glucose, this protein is critical in causing diabetic cardiomyopathy. In this study, we explored the molecular mechanism of glucose-induced TXNIP expression in cardiomyocytes. H9c2 rat cardiomyocytes were cultured at a low (5mM) or elevated (25mM) glucose concentration and in the presence or absence of a P38 MAP kinase inhibitor, to analyze whether or not the inhibitor will stop TXNIP from being upregulated. We found that the inhibitor caused a significant dose-dependent reduction of TXNIP suggesting that P38 MAP kinase signaling is involved in the regulation of cardiomyocyte TXNIP expression. Transient transfection studies are now under way to determine the TXNIP promoter region responsible for these effects.

Vitamin E supplementation as a strategy for preventing nosocomial infections

SIBS student – Morgan Jackson

SIBS mentor – Karen Iles

Pseudomonas aeruginosa infection is the most prevalent nosocomial infection in intubated patients, resulting in pulmonary edema which may culminate in lethal pneumonia. Several studies have established through in vitro and in vivo experiments that Vitamin E confers some protection against P. aeruginosa infection. Vitamin E is often termed an “antioxidant”, but its protective effects in P. aeruginosa infection extend beyond those of a simple antioxidant. The mechanisms whereby Vitamin E impacts the P. aeruginosa infectivity remain unclear.

Vitamin E may have a dual effect on the infection cascade. We hypothesize that it negatively impacts the ability of P. aeruginosa to invade the cell. Vitamin E may also exert a protective effect by enhancing the cell's antioxidant system as a Phase II gene inducer.

In vivo and in vitro model systems will be used to determine the effects of Vitamin E treatment on P. aeruginosa (K-strain, PAK) infectivity. With our collaborators, mice will be treated with Vitamin E or vehicle 18 hr before the instillation of 5 X10CFU of PAK and differences in survival will be recorded. MTT assays will be used in order to determine the effect of Vitamin E pretreatment on the viability of rat micro-vascular endothelial cells infected with PAK. Time courses (6-24 hrs) will be performed to determine if pretreatment with Vitamin E increases expression of several cytoprotective proteins (Western Blotting). Shorter time course incubations (0-6 hrs) with PAK will be performed in order to identify which cell signaling pathways are involved.

GSK3β inhibition protects healthy cells by repairing irradiation induced DNA damage

SIBS student - Tanu Patel
SIBS mentor - Eddy S. Yang

Cranial irradiation (IR) therapy often results in eventual neurocognitive deficits in memory and learning, especially for pediatric patients. These effects may be due to IR induced double-strand breaks (DSBs) in DNA in hippocampal neurons. DNA damage has been found to increase the amount of GSK3β expressed in normal irradiated cells. This accumulation of GSK3β ultimately leads to an increased amount of cell apoptosis. It has been found that inhibition of GSK3β increases DSB repair, and consequently decreases cell death in hippocampal neuronal cells but does not inhibit cell death in glioma cells. (Yang, 2011) This specificity in protection makes GSK3β inhibition an ideal therapeutic strategy for neuroprotection. In order to further understand the universality of the role that GSK3β inhibition plays in DNA repair mediated protection, we looked at this phenomenon in kidney cells. Our preliminary results suggest that there is indeed a protective effect on cells treated with GSK3β as opposed to cells treated with a control. However, further study is needed to confirm these preliminary results. Additionally, it has been hypothesized that the DNA repair and consequent cell survival seen with the inhibition of GSK3β is related to the subcellular localization of BRCA1. BRCA1 assists in non-homologous end joining (NHEJ) repair of DSBs if localized in the nucleus. However, radiation causes BRCA1 to shuttle from the nucleus to cytoplasm and results in cell apoptosis. (Yang, 2010) The results from our experiments testing the link between GSK3β and BRCA1 are pending.

Inhibition of the mTOR/PI3K Pathways to Enhance Sensitivity of Ovarian Cancer Cells to Chemotherapy Treatment

SIBS student: Lindy Pence

SIBS mentor: Charles Landen, M.D.

The American Cancer Society estimated for 2012 that 15,500 women in the United States would lose their lives to ovarian cancer. In addition, the 5-year survival rate for ovarian cancer is a disheartening 46%. This low rate is the result of ovarian cancer being frequently diagnosed at advanced stage, and having a high rate of recurrence and development of chemoresistance. To improve survival in ovarian cancer, many biologic therapies have been developed, but have shown disappointing results when used alone. Therefore this study investigates whether biologic pathways can be targeted in combination with chemotherapy in order to kill the resistant population that survives after primary therapy. The mTOR and PI3K pathways have been recognized as being frequently overactive in ovarian cancer cells, and these two pathways have been targeted individually with inhibitors. However, the redundancy and crosstalk between these pathways have limited the success of single-target inhibitors. Recently, inhibitors have been developed that concurrently target both mTOR and PI3K, potentially eliminating this feedback loop.

In this study one such inhibitor, PF-04691502, was tested on both chemoresistant and chemosensitive ovarian cancer cell lines for decreasing cell viability with and without the presence of chemotherapy agents paclitaxol and carboplatin. The chemosensitive cell lines are highly sensitive to the dual inhibitor, but no synergy was seen when combined with chemotherapy. Chemoresistant lines are less sensitive to PF-04691502 alone. Whether PF-04691502 sensitizes these resistant cells to chemotherapy is pending. Thus far PF-04691502 appears to be a promising agent in chemosensitive ovarian cancer. 

Making New Photoreceptors from Retinal Pigment Epithelium

SIBS student:  Rachael Sarrett

SIBS mentors: Timothy Kraft, Run-Tao Yan and Shu-Zhen Wang

The retina contains photoreceptors that transform light into electrical signals that are passed on to, and interpreted by the brain. The two types, rods and cones, mediate night vision and daytime vision respectively. Mammalian photoreceptors cannot regenerate, thus photoreceptor death can permanently impair vision, even leading to complete blindness. This project aims to identify a viable source and feasible means of generating de novo, functional photoreceptors for replacement therapy. We propose that the retinal pigment epithelium (RPE), a pigmented layer of tissue located directly behind the photoreceptors can be stimulated to differentiate into photoreceptor cells genetically.

Transgenic animals were examined for the presence of new cells, the morphology of the new cells, and for cell specific protein markers identifying them as photoreceptors. We used immunohistochemistry to ensure that these newly created cells contained key cone proteins. Then, using an electroretinogram (ERG), we tested the photoreceptors in these transgenic mice to determine whether or not the extra layer of photoreceptors functions properly (i.e. creates an electric response in response to light) and compared these results to wild-type mice. The ERG measures the summed electrical response of all retinal cells responding to a change in light. Photoreceptor cells’ electrical response to light, the negative a-wave of the ERG, was disproportionately larger in ngn-3 transgenic animals under both light- and dark-adapted conditions suggesting that the new cells formed were indeed functional photoreceptors.

Investigation of the Mechanism of Regulation of Proliferation and Radiation Sensitivity by the MARCKS Protein in Glioma Cells.

SIBS student - Samantha Scanlon,

SIBS mentors - John S. Jarboe and Christopher D. Willey

Glioblastoma multiforme (GBM) represents the most common and deadly form of glioma, with the median post-diagnosis survival being 12 months. In xenograft models, GBM has been shown to be more radiation resistant when the phosphatidylinositol-3-kinase (PI3K)/Akt pathway is active, since it promotes increased cell growth, DNA damage repair, and survival. In this pathway, phosphatidyl inositol bisphosphate (PIP2) is converted to the triphosphate (PIP3) by PI3K, which leads to Akt activation. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) is a potential regulator for the availability of PIP2 to the PI3K/Akt pathway as it is capable of sequestering PIP2 at the membrane via electrostatic interactions. Our objective is to further elucidate the mechanism of this regulation, which is anticipated to occur through a reversible sequestration of PIP2 at the membrane of the cell by MARCKS. This will be investigated through the over-expression of various MARCKS mutants in three glioma cell lines (U87, U373, and U251) and subsequent observation of its effects on the PI3K/Akt pathway, proliferation, radiation sensitivity, DNA damage repair, and apoptosis.

The role of ERK phosphorylation in BQCA-mediated LTD in CA1 hippocampal neurons

SIBS student - Paige Souder

SIBS Mentors - Robert Mans and Lori McMahon

Therapeutic targets for Alzheimer’s disease (AD) are currently being studied in various aspects.  One such target, the M1 muscarinic G-protein coupled acetylcholine receptor (mAChR), has been found in previous studies to modulate multiple hallmarks of AD pathology.  Agonists of the M1 mAChR decrease cognitive deficits as well as tau and Aβ aggregation.   A novel pharmaceutical agent, 3-(4-bromobenzoyl)-2-quinolinecarboxaldehyde (BQCA), acts as an allosteric potentiator of M1 and has been found to successfully reverse AD pathology in mice.  Further studies of BQCA have found it induces long-term depression (LTD), a vital form of synaptic plasticity, in rat hippocampal slices.  The current study investigates the intracellular signaling events mediating BQCA-induced LTD in the CA1 subfield of the hippocampus, a region necessary for normal learning and memory.  M1 receptor activation is known to trigger phosphorylation of extracellular-signal-regulated kinase 1/2 (ERK 1/2) Here we test the prediction that a 15 min treatment with BQCA, a duration known to induce LTD in CA1, will increase the amount of phosphorylated (activated) ERK in CA1 synapses. 

Extra Ribosomal Functions of RPS25

SIBS student - Carly Twarog

SIBS mentor - Sunnie Thompson

The vast majority of mRNAs are translated through a cap-dependent mechanism that requires a 5’ cap and 10-13 initiation factors in order to recruit the 40S ribosomal subunit. However, some viral and cellular mRNAs use an alternative mechanism of initiating translation that requires an internal ribosome entry site (IRES) in the 5’ untranslated region (UTR). Ribosomal protein S25 (Rps25) is required for IRES translation, but not for cap-dependent translation, ribosome biogenesis, or ribosome function. Thus, it is reasonable to assume usual cellular processes could proceed in the absence of Rps25. However, previous studies have shown that ribosomal proteins can have specialized functions apart from their function on the ribosome. Since Rps25 is located on the surface of the 40S ribosome it may be able to associate or disassociate from the ribosome under certain cellular conditions. The goal of our study is to determine if there are any ribosomal free copies of Rps25 in the cell. Polysomes will be separated on a sucrose density gradient, fractionated, and protein will be looked at by Western Blot Analysis to determine where Rps25 associates. Considering some viruses, such as the Hepatitis C virus, require host Rps25 for translation of their genome, it is possible that Rps25 could be a target for drug or gene therapy if proven not to play a vital role in host cells.