Specific Role for GSK3α in Limiting Long-Term Potentiation in CA1 Pyramidal Neurons of Adult Mouse Hippocampus.

Glycogen synthase kinase-3 (GSK3) mediates phosphorylation of several hundred proteins, and its aberrant activity is associated with an array of prevalent disorders. The two paralogs, GSK3α and GSK3ß, are expressed ubiquitously and fulfill common as well as unique tasks throughout the body. In the CNS, it is established that GSK3 is involved in synaptic plasticity. However, the relative roles of GSK3 paralogs in synaptic plasticity remains controversial. Here, we used hippocampal slices obtained from adult mice to determine the role of each paralog in CA3-CA1 long-term potentiation (LTP) of synaptic transmission, a form of plasticity critically required in learning and memory. Conditional Camk2a Cre-driven neuronal deletion of the Gsk3a gene, but not Gsk3b, resulted in enhanced LTP. There were no changes in basal synaptic function in either of the paralog-specific knockouts, including several measures of presynaptic function. Therefore, GSK3α has a specific role in serving to limit LTP in adult CA1, a postsynaptic function that is not compensated by GSK3ß.

A Low-Therapeutic Dose of Lithium Inhibits GSK3 and Enhances Myoblast Fusion in C2C12 Cells.

Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/ß-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3ß) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5-1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3ß (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2-2.5 fold, p < 0.05), a result associated with an increase in total ß-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (-86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions.

Identification of CDC25 as a Common Therapeutic Target for Triple-Negative Breast Cancer.

CDK4/6 inhibitors are effective against cancer cells expressing the tumor suppressor RB1, but not RB1-deficient cells, posing the challenge of how to target RB1 loss. In triple-negative breast cancer (TNBC), RB1 and PTEN are frequently inactivated together with TP53. We performed kinome/phosphatase inhibitor screens on primary mouse Rb/p53-, Pten/p53-, and human RB1/PTEN/TP53-deficient TNBC cell lines and identified CDC25 phosphatase as a common target. Pharmacological or genetic inhibition of CDC25 suppressed growth of RB1-deficient TNBC cells that are resistant to combined CDK4/6 plus CDK2 inhibition. Minimal cooperation was observed in vitro between CDC25 antagonists and CDK1, CDK2, or CDK4/6 inhibitors, but strong synergy with WEE1 inhibition was apparent. In accordance with increased PI3K signaling following long-term CDC25 inhibition, CDC25 and PI3K inhibitors effectively synergized to suppress TNBC growth both in vitro and in xenotransplantation models. These results provide a rationale for the development of CDC25-based therapies for diverse RB1/PTEN/TP53-deficient and -proficient TNBCs.

A ZIP6-ZIP10 heteromer controls NCAM1 phosphorylation and integration into focal adhesion complexes during epithelial-to-mesenchymal transition.

The prion protein (PrP) evolved from the subbranch of ZIP metal ion transporters comprising ZIPs 5, 6 and 10, raising the prospect that the study of these ZIPs may reveal insights relevant for understanding the function of PrP. Building on data which suggested PrP and ZIP6 are critical during epithelial-to-mesenchymal transition (EMT), we investigated ZIP6 in an EMT paradigm using ZIP6 knockout cells, mass spectrometry and bioinformatic methods. Reminiscent of PrP, ZIP6 levels are five-fold upregulated during EMT and the protein forms a complex with NCAM1. ZIP6 also interacts with ZIP10 and the two ZIP transporters exhibit interdependency during their expression. ZIP6 contributes to the integration of NCAM1 in focal adhesion complexes but, unlike cells lacking PrP, ZIP6 deficiency does not abolish polysialylation of NCAM1. Instead, ZIP6 mediates phosphorylation of NCAM1 on a cluster of cytosolic acceptor sites. Substrate consensus motif features and in vitro phosphorylation data point toward GSK3 as the kinase responsible, and interface mapping experiments identified histidine-rich cytoplasmic loops within the ZIP6/ZIP10 heteromer as a novel scaffold for GSK3 binding. Our data suggests that PrP and ZIP6 inherited the ability to interact with NCAM1 from their common ZIP ancestors but have since diverged to control distinct posttranslational modifications of NCAM1.

Towards the preparation of radiolabeled 1-aryl-3-benzyl ureas: Radiosynthesis of [(11)C-carbonyl] AR-A014418 by [(11)C]CO(2) fixation.

The highly selective glycogen synthase kinase-3 (GSK-3) inhibitor N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) was radiolabeled with carbon-11 ((11)C; half-life=20.4min) at the urea moiety via [(11)C]CO(2) fixation. Reaction of [(11)C]CO(2) with 4-methoxybenzylamine in the presence of a CO(2) fixating base was followed by dehydration with POCl(3) and addition of 2-amino-5-nitrothiazole to prepare [(11)C-carbonyl] AR-A014418. This reaction resulted in an 8% uncorrected radiochemical yield, based on [(11)C]CO(2), with high specific activity (4Ci/µmol) within 30min. An in vitro GSK-3ß enzyme activity assay revealed that AR-A014418 (K(i)=770nM) is not as potent as previously claimed. The [(11)C]CO(2) fixation methodology described herein should prove generally applicable to preparing 1-aryl-3-benzyl-[(11)C-carbonyl] ureas as radiotracers for positron emission tomography.

Rationally designed PKA inhibitors for positron emission tomography: Synthesis and cerebral biodistribution of N-(2-(4-bromocinnamylamino)ethyl)-N-[11C]methyl-isoquinoline-5-sulfonamide.

Protein kinase A (PKA) is an important signal transduction target for drug development because it influences critical cellular processes implicated in neuropsychiatric illnesses such as major depressive disorder. The goal of the present study was to develop the first imaging agent for measuring the levels of PKA with positron emission tomography (PET). By rational derivatization of 5-isoquinoline sulfonamides, it was found that the introduction of a methyl group to the sulphonamidic nitrogen on the known PKA inhibitors N-(2-aminoethyl)isoquinoline-5-sulfonamide (H-9, 1) and N-(2-(4-bromocinnamylamino)ethyl)isoquinoline-5-sulfonamide (H-89, 2), (yielding N-(2-aminoethyl)-N-methyl-isoquinoline-5-sulfonamide (4) and N-(2-(4-bromocinnamylamino)ethyl)-N-methyl-isoquinoline-5-sulfonamide (5), respectively) does not appreciably reduce in vitro potency toward PKA. We have facilitated the synthesis of 4 by reacting isoquinoline-5-sulfonyl chloride with N-methylethylenediamine (20% yield). Several techniques were used to thoroughly characterize 4 including multi ((1)H, (13)C and (15)N) NMR spectroscopy and X-ray crystallography. Compound 4 and 1-(4-bromophenyl)-1-propen-3-yl bromide were reacted to produce 5 in 16% yield. Compound 2 was reacted with [(11)C]CH(3)I to prepare N-(2-(4-bromocinnamylamino) ethyl)-N-[(11)C]methyl-isoquinoline-5-sulfonamide ([(11)C]5), with a decay-corrected radiochemical yield of 32%, based on [(11)C]CO(2). [(11)C]5 was produced with >98% radiochemical purity and 1130mCi/mumol specific activity after 40min (end of synthesis). Conscious rats were administered [(11)C] 5 and sacrificed at 5, 15, 30 and 60min after injection. Radioactivity from all excised brain regions was <0.2%ID/g at all time points. The modest brain penetration of [(11)C]5 may limit its use for studying PKA in the central nervous system.