Browsing by Subject "AMPK"
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Item Regulation and Function of the Phosphatase PHLPP2 in Leukemia(2017-08) Yan, YanPHLPP2, a member of the PHLPP phosphatase family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little was known, however, regarding its regulation and function in hematological malignancies. The first half of this dissertation describes a novel miR-17~92-based mechanism for repression of PHLPP2 protein expression in late differentiation stage acute myeloid leukemia (AML) subtypes. ATRA (all-trans retinoic acid), a drug used for terminally differentiating AML subtypes, was able to induce PHLPP2 protein levels and phosphatase activity significantly by suppressing miR-17-92 expression. The effect of ATRA on miR-17~92 expression was mediated through its target, transcription factor C/EBP, which interacts with the intronic promoter of the miR-17~92 gene cluster to inhibit its transactivation. The second half of this dissertation provides evidence for a novel metabolic function for PHLPP2 and describes the first identification of the energy sensing kinase, AMPK, as a unique PHLPP2 substrate. PHLPP2 could dephosphorylate phospho-AMPK (T172) both intracellularly and in vitro. PHLPP2 silencing protected Jurkat T-ALL cells from an apoptotic response to low glucose-induced metabolic stress through activation of AMPK signaling. The pro-survival effect of PHLPP2 knockdown under metabolic stress is likely mediated through AMPK-activated fatty acid oxidation. PHLPP2 regulates AMPK phosphorylation in a variety of tumor types and is the first specific AMPK phosphatase to be identified. These studies on PHLPP2 expression and function expand current knowledge and understanding of the role of PHLPP phosphatases in cancer, and particularly in leukemia. In light of the pivotal role played by AMPK in a number of metabolic diseases, the PHLPP2/AMPK axis is also expected to provide new insights into therapies targeting these diseases.Item The role of astrocytic adenosine monophosphate-activated protein kinase in cocaine-related behaviors(2022-01) Buczek, LauraCocaine use induces changes in the brain’s structure and function inducing long-lasting changes in behavior and cognition. There are no FDA approved treatments for cocaine use disorder. Investigating cocaine-induced neuroadaptations, specifically changes in brain protein expression and function, will inform molecular targets for future therapeutic drug development. Two proteins, glial glutamate transporter 1 (GLT-1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) are downregulated upon cocaine exposure. It is unknown whether AMPK, expressed in neurons and astrocytes, regulates GLT-1 in the striatum in a cell-specific manner. I will first determine how astrocyte-specific manipulation of striatal AMPK impacts GLT-1 expression and function in the striatum. I will then determine whether the same astrocytic AMPK manipulations influence cocaine locomotor sensitization and acquisition of cocaine self-administration.Item Verification of AMPK Knockdown in Sprague Dawley Rats using a Vivo-Morpholino Antisense Oligonucleotide(2024-01-09) Hunsader, Peter; Hernandez, Edith; Moore, Maddi; Slosky, Lauren; Spencer, SadeMetformin, a drug typically used to treat type II diabetes, can decrease cue-induced cocaine seeking in rats. The proposed mechanism of action for this effect is the activation of adenosine monophosphate-activated protein kinase (AMPK). To confirm this mechanism for the effect of metformin we are validating a technique for knocking down AMPK in the nucleus accumbens core (NAcC) in vivo. This study tests whether microinjections of a vivo-morpholino antisense oligonucleotide (VM-ASO) in the NAcC is an effective method of knocking down AMPK in Sprague Dawley rats. VM-ASO binds target mRNA and prevents translation. This experiment used 8 Sprague Dawley rats (6 male, 2 female). Initially, rats underwent stereotaxic intracranial surgery for the insertion of a cannula into both hemispheres of the NAcC (from bregma: AP +1.5; ML: +/- 1.8; DV: - 5.5mm). Then, 140 pmol of AMPK VM-ASO or scrambled control nucleotide was microinjected in each hemisphere at a rate of 0.2 µl/minute 2mm below the base of the cannula. After a one-minute diffusion period, the microinjectors were removed. This process was repeated for four days. Eight days after the last microinjection, tissue samples were collected and AMPK levels were analyzed via western blot. It was found that microinjections of AMPK VM-ASO did not significantly decrease AMPK levels in the NAcC.