Ulrike Winkler

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 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.

Genetic Deletion of Laminin Isoforms β2 and γ3 Induces a Reduction in Kir4.1 and Aquaporin-4 Expression and Function in the Retina

Background Glial cells such as retinal Müller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Müller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Müller cells. Methodology/Principal Findings The aim of the present study was to test the function of the β2 and γ3 containing laminins in murine Müller cells. We used knockout mice with genetic deletion of both β2 and γ3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a down-regulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Müller cells from laminin β2 and γ3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice. Conclusion These findings demonstrate a strong impact of laminin β2 and γ3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Müller cells.

The Vinculin-ΔIn20/21 Mouse: Characteristics of a Constitutive, Actin-Binding Deficient Splice Variant of Vinculin

Background The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models. Methodology/Principal Findings Here, we investigate vinculin-ΔEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-ΔEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-ΔIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-ΔIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-ΔEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-ΔEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-ΔEx20 variant unveils vinculins dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites. Conclusions/Significance Vinculin-ΔEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development.

Monitoring ATP dynamics in electrically active white matter tracts

In several neurodegenerative diseases and myelin disorders, the degeneration profiles of myelinated axons are compatible with underlying energy deficits. However, it is presently impossible to measure selectively axonal ATP levels in the electrically active nervous system. We combined transgenic expression of an ATP-sensor in neurons of mice with confocal FRET imaging and electrophysiological recordings of acutely isolated optic nerves. This allowed us to monitor dynamic changes and activity-dependent axonal ATP homeostasis at the cellular level and in real time. We find that changes in ATP levels correlate well with compound action potentials. However, this correlation is disrupted when metabolism of lactate is inhibited, suggesting that axonal glycolysis products are not sufficient to maintain mitochondrial energy metabolism of electrically active axons. The combined monitoring of cellular ATP and electrical activity is a novel tool to study neuronal and glial energy metabolism in normal physiology and in models of neurodegenerative disorders. DOI: http://dx.doi.org/10.7554/eLife.24241.001

Crosstalk of Signaling and Metabolism Mediated by the NAD + /NADH Redox State in Brain Cells

The energy metabolism of the brain has to be precisely adjusted to activity to cope with the organ’s energy demand, implying that signaling regulates metabolism and metabolic states feedback to signaling. The NAD+/NADH redox state constitutes a metabolic node well suited for integration of metabolic and signaling events. It is affected by flux through metabolic pathways within a cell, but also by the metabolic state of neighboring cells, for example by lactate transferred between cells. Furthermore, signaling events both in neurons and astrocytes have been reported to change the NAD+/NADH redox state. Vice versa, a number of signaling events like astroglial Ca2+ signals, neuronal NMDA-receptors as well as the activity of transcription factors are modulated by the NAD+/NADH redox state. In this short review, this bidirectional interdependence of signaling and metabolism involving the NAD+/NADH redox state as well as its potential relevance for the physiology of the brain and the whole organism in respect to blood glucose regulation and body weight control are discussed.

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.

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.

Activity-dependent modulation of intracellular ATP in cultured cortical astrocytes

Brain function is absolutely dependent on an appropriate supply of energy. A shortfall in supply—as occurs, for instance, following stroke—can lead rapidly to irreversible damage to this vital organ. While the consequences of pathophysiological energy depletion have been well documented, much less is known about the physiological energy dynamics of brain cells, although changes in the intracellular concentration of adenosine triphosphate (ATP), the major energy carrier of cells, have been postulated to contribute to cellular signaling. To address this issue more closely, we have investigated intracellular ATP in cultured primary cortical astrocytes by time‐lapse microscopy using a genetically encoded fluorescent sensor for ATP. The cytosolic ATP sensor signal decreased after application of the neurotransmitter glutamate in a manner dependent on both glutamate concentration and glutamate transporter activity, but independent of glutamate receptors. The application of dopamine did not affect ATP levels within astrocytes. These results confirm that intracellular ATP levels in astrocytes do indeed respond to changes in physiological activity and pave the way for further studies addressing factors that affect regulation of ATP.