Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny

Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms

Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation

ProSAP/Shank are scaffolding proteins that localize to the postsynaptic density (PSD). This study shows that Zn2+ ions directly regulate the localization and recruitment of Shank/ProSAP1/2 to PSDs to facilitate synapse formation and maturation

Heterogeneous Nuclear Ribonucleoprotein K Interacts with Abi-1 at Postsynaptic Sites and Modulates Dendritic Spine Morphology

BACKGROUND: Abelson-interacting protein 1 (Abi-1) plays an important role for dendritic branching and synapse formation in the central nervous system. It is localized at the postsynaptic density (PSD) and rapidly translocates to the nucleus upon synaptic stimulation. At PSDs Abi-1 is in a complex with several other proteins including WASP/WAVE or cortactin thereby regulating the actin cytoskeleton via the Arp 2/3 complex. PRINCIPAL FINDINGS: We identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a 65 kDa ssDNA/RNA-binding-protein that is involved in multiple intracellular signaling cascades, as a binding partner of Abi-1 at postsynaptic sites. The interaction with the Abi-1 SH3 domain is mediated by the hnRNPK-interaction (KI) domain. We further show that during brain development, hnRNPK expression becomes more and more restricted to granule cells of the cerebellum and hippocampal neurons where it localizes in the cell nucleus as well as in the spine/dendritic compartment. The downregulation of hnRNPK in cultured hippocampal neurons by RNAi results in an enlarged dendritic tree and a significant increase in filopodia formation. This is accompanied by a decrease in the number of mature synapses. Both effects therefore mimic the neuronal morphology after downregulation of Abi-1 mRNA in neurons. CONCLUSIONS: Our findings demonstrate a novel interplay between hnRNPK and Abi-1 in the nucleus and at synaptic sites and show obvious similarities regarding both protein knockdown phenotypes. This indicates that hnRNPK and Abi-1 act synergistic in a multiprotein complex that regulates the crucial balance between filopodia formation and synaptic maturation in neurons

An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

BACKGROUND: The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell. PRINCIPAL FINDINGS: We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners. CONCLUSIONS: Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Small-Molecule XIAP Inhibitors Enhance γ-Irradiation-Induced Apoptosis in Glioblastoma12

Because evasion of apoptosis can cause radioresistance of glioblastoma, there is a need to design rational strategies that counter apoptosis resistance. In the present study, we investigated the potential of targeting the antiapoptotic protein XIAP for the radiosensitization of glioblastoma. Here, we report that small-molecule XIAP inhibitors significantly enhance γ-irradiation-induced loss of viability and apoptosis and cooperate with γ-irradiation to suppress clonogenic survival of glioblastoma cells. Analysis of molecular mechanisms reveals that XIAP inhibitors act in concert with γ-irradiation to cause mitochondrial outer membrane permeabilization, caspase activation, and caspase-dependent apoptosis. Importantly, XIAP inhibitors also sensitize primary cultured glioblastoma cells derived from surgical specimens as well as glioblastoma-initiating stemlike cancer stem cells for γ-irradiation. In contrast, they do not increase the toxicity of γ-irradiation on some nonmalignant cells of the central nervous system, including rat neurons or glial cells, pointing to some tumor selectivity. In conclusion, by demonstrating for the first time that small-molecule XIAP inhibitors increase the radiosensitivity of glioblastoma cells while sparing normal cells of the central nervous system, our findings build the rationale for further (pre)clinical development of XIAP inhibitors in combination with γ-irradiation in glioblastoma

Downregulation of hnRNPK mimics the “Abi-1 depletion phenotype” in neurons.

<p>(A) hnRNPK-RNAi knockdown in NIH3T3 cells with a construct targeting the 3′UTR-region of hnRNPK. (I) hnRNPK is endogenously expressed in NIH3T3, HeLA and Cos7 cells and is detectable in cultured hippocampal neurons (DIV21). (II) After transfection of NIH3T3 cells with an hnRNPK-RNAi construct for 3 days, the cells were fixed and stained with an antibody against hnRNPK. Only untransfected cells in close proximity to the RNAi-transfected cell in the upper right are immunopositive for hnRNPK, with a predominant distribution of the protein in the nucleus. (III) After transfection of NIH3T3 cells with an hnRNPK-RNAi construct for 3 days, protein expression of hnRNPK is markedly suppressed as confirmed by Western blotting compared to vector control transfected cells. (IV) Double transfections using two different hnRNPK-RNAi constructs, one targeting the 3′UTR of the hnRNPK sequence and one targeting the coding sequence of hnRNPK together with an hnRNPK-Myc-construct which is resistant against RNAi due to 4 nucleotide exchanges in the RNA leading to an unaltered amino acid sequence. The staining against the Myc-tag shows a decreased protein level solely when using the non-resistant construct together with the RNAi targeting the CDS (coding sequence). (B) Neuronal transfection of Abi-1-RNAi and hnRNPK-RNAi constructs. (I) In contrast to the control vector (pSuper), transfection of RNAi constructs resulted in an obvious change of neuronal morphology. The downregulation of hnRNPK as well as Abi-1 is leading to an extended and extremely branched dendritic tree. (II) The number of branching points within the dendritic compartment is significantly upregulated in both RNAi groups compared to the control transfection. (III) The analysis of the dendritic tree shows a significant shift of dendrites towards small, filopodia-like tertiary dendrites. Scale bars are as indicated.</p