Franziska Wilhelm

Split-CreERT2: Temporal Control of DNA Recombination Mediated by Split-Cre Protein Fragment Complementation

Background DNA recombination technologies such as the Cre/LoxP system advance modern biological research by allowing conditional gene regulation in vivo. However, the precise targeting of a particular cell type at a given time point has remained challenging since spatial specificity has so far depended exclusively on the promoter driving Cre recombinase expression. We have recently established split-Cre that allows DNA recombination to be controlled by coincidental activity of two promoters, thereby increasing spatial specificity of Cre-mediated DNA recombination. To allow temporal control of split-Cre-mediated DNA recombination we have now extended split-Cre by fusing split-Cre proteins with the tamoxifen inducible ERT2 domain derived from CreERT2. Methodology/Principal Findings In the split-CreERT2 system, Cre-mediated DNA recombination is controlled by two expression cassettes as well as the time of tamoxifen application. By using two independent Cre-dependent reporters in cultured cells, the combination of NCre-ERT2+ERT2-CCre was identified as having the most favorable properties of all constructs tested, showing an induction ratio of about 10 and EC50-values for 4-hydroxy-tamoxifen of 10 nM to 70 nM. Conclusions/Significance These characteristics of split-CreERT2 in vitro indicate that split-CreERT2 will be well suited for inducing DNA recombination in living mice harboring LoxP-flanked alleles. In this way, split-CreERT2 will provide a new tool of modern genetics allowing spatial and temporal precise genetic access to cell populations defined by the simultaneous activity of two promoters.

The NAD+/NADH redox state in astrocytes: Independent control of the NAD+ and NADH content

The intracellular redox state is established by several redox pairs, such as NAD+/NADH and NADP+/NADPH and glutathione. This redox state is a crucial determinant of cellular metabolism and function. Astrocytes are an important cell population contributing to brain metabolism and brain energy supply, so a careful control of these redox pairs is essential for proper brain function. Despite this, little is known about control of the NAD+ and NADH content within the brain or in astrocytes. Therefore, we here analyzed the NAD+ and NADH content of mouse tissue and cultured cortical astrocytes. The NAD+/NADH ratio increased in most tissues during development from newborn to adult mice. The basal redox ratio of cultured astrocytes was about 3.8 and similar to the redox ratio of the cortex of newborn mice. Although the NADH content of these cells was highly sensitive to the concentration of energy substrates and to modulation of energy metabolism, the NAD+ content was surprisingly constant under these conditions. In contrast, application of nicotine amide or nicotinamide mononucleotide, which are precursors for NAD+ biosynthesis, slowly increased NAD+ content while leaving NADH levels unaffected. Finally, inhibiting the NAD+‐degrading enzyme poly‐(ADP‐ribose)‐polymerase increased NAD+ content slightly without affecting NADH levels, whereas inhibition of sirtuins had no effect. These results indicate that, in addition to converting NAD+ to NADH and vice versa during redox reactions, the content of both partners of this redox pair is additionally controlled by other mechanisms.

The biphasic NAD(P)H fluorescence response of astrocytes to dopamine reflects the metabolic actions of oxidative phosphorylation and glycolysis.

J. Neurochem. (2010) 115, 483–492.

Ca²⁺ signals of astrocytes are modulated by the NAD⁺/NADH redox state.

J. Neurochem. (2012) 120, 1014–1025.

ABCG2 expression is correlated neither to side population nor to hematopoietic progenitor function in human umbilical cord blood

OBJECTIVE The ABCG2 transporter has been identified as a determinant of the side population (SP) in murine bone marrow and a potential marker for primitive stem cells. To assess the potential of the SP phenotype and ABCG2 expression to identify stem cells in human umbilical cord blood (hUCB), we have examined directly the relationship between SP; expression of ABCG2, CD133, and CD34; and hematopoietic potential in UCB samples. MATERIALS AND METHODS Multicolor fluorescence activated cell sorting analysis was combined with the Hoechst SP procedure to allow the simultaneous detection of the SP phenotype together with surface markers in cells from fresh and cryopreserved UCB. Sorted populations were analyzed for cobblestone area-forming cell (CAFC) activity and by quantitative reverse transcriptase polymerase chain reaction for expression of mRNA from the ABC transporters ABCG2, MDR1, and MRP1. ABCG2(+) cells were enriched by magnetic-activated cell sorting for stringent analysis. RESULTS hUCB-derived SP cells were negative for ABCG2, but comprise approximately 20% CD133(+)/CD34(+) cells. Sorted SP cells from UCB were enriched 20-fold for week 13 CAFC activity, while magnetic-activated cell sorting-enriched ABCG2(+) cells retained no hematopoietic activity either in CAFC or liquid cultures. There was no significant difference in the SP frequency, immunophenotype, or CAFC potential of fresh and cryopreserved UCB. ABCG2 mRNA was not enriched in the SP and was specifically diminished ninefold in CD133 cells, which were eightfold enriched for MDR1 mRNA. CONCLUSION We find no evidence for an association of ABCG2 with SP activity or hematopoietic progenitor function in hUCB.

Sirolimus treatment of severe PTEN hamartoma tumor syndrome: case report and in vitro studies

Background:Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is caused by germ line mutations in the PTEN gene. Symptoms include cancer predisposition, immune deviations, and lipomas/lipomatosis. No causal standard therapy is available. We describe a therapeutic attempt with the mammalian target of rapamycin (mTOR) inhibitor sirolimus for a PHTS patient suffering from thymus hyperplasia and lipomatosis. We furthermore assessed the in vitro effects of sirolimus and other inhibitors on lipoma cells of the patient.Methods:The patient underwent clinical and blood examinations and whole-body magnetic resonance imaging to assess tumor sizes. Lipoma cells of the patient were incubated with inhibitors of the phosphoinositide-3-kinase (PI3K)/AKT/mTOR signaling pathway to analyze the effects on proliferation, adipocyte differentiation, and survival in vitro.Results:Sirolimus treatment improved somatic growth and reduced thymus volume. These effects diminished over the treatment period of 19 mo. Sirolimus decreased lipoma cell proliferation and adipocyte differentiation in vitro but did not cause apoptosis. PI3K and AKT inhibitors induced apoptosis significantly.Conclusion:Sirolimus treatment led to an improvement of the patient’s clinical status and a transient reduction of the thymus. Our in vitro findings point to PI3K and AKT inhibitors as potential treatment options for patients with severe forms of PHTS.

Multifunctional roles of NAD+ and NADH in astrocytes

The control and maintenance of the intracellular redox state is an essential task for cells and organisms. NAD+ and NADH constitute a redox pair crucially involved in cellular metabolism as a cofactor for many dehydrogenases. In addition, NAD+ is used as a substrate independent of its redox-carrier function by enzymes like poly(ADP)ribose polymerases, sirtuins and glycohydrolases like CD38. The activity of these enzymes affects the intracellular pool of NAD+ and depends in turn on the availability of NAD+. In addition, both NAD+ and NADH as well as the NAD+/NADH redox ratio can modulate gene expression and Ca2+ signals. Therefore, the NAD+/NADH redox state constitutes an important metabolic node involved in the control of many cellular events ranging from the regulation of metabolic fluxes to cell fate decisions and the control of cell death. This review summarizes the different functions of NAD+ and NADH with a focus on astrocytes, a pivotal glial cell type contributing to brain metabolism and signaling.

The human ubiquitin C promoter drives selective expression in principal neurons in the brain of a transgenic mouse line

The specificity of promoters used to drive the expression of proteins of interest is a crucial determinant of transgenesis. Numerous strategies have been developed to restrict expression on a certain cell population. On the other hand it has also remained challenging to obtain ubiquitous expression of transgenes which is needed for example to generate recombination reporter mice or to induce expression by recombination mediated excision of STOP-cassettes. We have generated transgenic mice with the expression of nuclear β-galactosidase driven by the human ubiquitin C promoter thought to mediate ubiquitous expression. However, in the brains of these transgenic mice the expression of the transgene was strikingly limited to principal neurons, while no expression was detected in interneurons or glial cells. These results indicate that the human ubiquitin C promoter might be useful to selectively target projections neurons of the brain.

Deletion of the cell adhesion adaptor protein vinculin disturbs the localization of GFAP in Bergmann glial cells

Astrocytes operate in close spatial relationship to other cells including neurons. Structural interaction is controlled by a dynamic interplay between actin‐based cell motility and contact formation via cell–cell and cell–extracellular matrix adhesions. A central player in the control of cell adhesion is the cytoskeletal adaptor protein Vinculin. Incorporation of Vinculin affects mechanical properties and turnover of cell adhesion sites. To study the in vivo function of Vinculin in astrocytes, a mouse line with astrocyte specific and inducible deletion of vinculin was generated. Deletion of vinculin decreased the expression of the glial acidic fibrillary protein (GFAP) in Bergmann glial cells in the cerebellum. In addition, localization of GFAP to Bergmann glial endfeet was disturbed, indicating a role for vinculin in controlling its expression and localization. In contrast, vimentin expression, morphology, activation state and polarity of the targeted cells as well as the localization of the extracellular matrix protein laminin was not compromised. Furthermore, stab wound lesions were performed in the cerebellar cortex. In both wildtype and vinculin knockout mice GFAP expression was upregulated in Bergmann glial cells of the lesioned area with no differences observed between genotypes in expression and localization of GFAP. These results propose a selective requirement for vinculin in cellular events related to cell adhesion in vivo. As in vitro data suggested a major role for vinculin in the control of the cytoskeletal connection affecting mechanical stability and cell motility, our data add a note of caution to the extrapolation of in vitro data to in vivo function.

Phosphatidylinositol 3-kinase (PI3K) signalling regulates insulin-like-growth factor binding protein-2 (IGFBP-2) production in human adipocytes

OBJECTIVE Insulin-like-growth factor binding protein 2 (IGFBP-2) is thought to be a marker for the phosphatase and tensin homolog (PTEN) status and activity of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. We aimed to evaluate whether or not lipoma cells of a patient with a heterozygous deletion in the PTEN gene would produce more IGFBP-2 than PTEN non deficient control cells. Moreover, we analysed the influence of pharmacological inhibitors of the PI3K/AKT/mTOR pathway on IGFBP-2 production. DESIGN PTEN deficient preadipocytes and control PTEN non deficient preadipocytes were differentiated in vitro and treated with the respective inhibitors. PTEN was transiently down regulated by siRNA in human preadipocytes. IGFBP-2 mRNA and protein expression and IGFBP-2 in culture supernatant were measured. RESULTS PTEN deficient lipoma cells were found to produce IGFBP-2 during in vitro differentiation in comparable amounts to PTEN non deficient cells. In contrast, acute down regulation of PTEN in preadipocytes resulted in enhanced production of IGFBP-2. Incubation with the PI3K inhibitors LY294002 and wortmannin decreased IGFBP-2 mRNA and protein. Neither the mTOR complex 1 inhibitor rapamycin nor PD98059, an inhibitor of MEK (mitogen-activated protein kinase kinase), showed a significant effect on IGFBP-2 production. CONCLUSION IGFBP-2 production in PTEN deficient preadipocytes was not influenced by PTEN deficiency or by inhibition of mTORC1 and MAPK. In contrast, inhibition of PI3K decreased IGFBP-2 expression and secretion.