Petra G. Hirrlinger

Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2

Inducible gene modification using the Cre/loxP system provides a valuable tool for the analysis of gene function in the active animal. GFAP‐Cre transgenic mice have been developed to achieve gene recombination in astrocytes, the most abundant cells of the central nervous system, with pivotal roles during brain function and pathology. Unfortunately, these mice displayed neuronal recombination as well, since the GFAP promoter is also active in embryonic radial glia, which possess a substantial neurogenic potential. To enable the temporal control of gene deletions in astrocytes only, we generated a transgenic mouse with expression of CreERT2, a fusion protein of the DNA recombinase Cre and a mutated ligand‐binding domain of the estrogen receptor, under the control of the human GFAP promoter. In offspring originating from crossbreedings of GFAP‐CreERT2‐transgenic mice with various Cre‐sensitive reporter mice, consecutive intraperitoneal injections of tamoxifen induced genomic recombination selectively in astrocytes of almost all brain regions. In Bergmann glia, which represent the main astroglial cell population of the cerebellum, virtually all cells showed successful gene recombination. When adult mice received cortical stab wound lesions, simultaneously given tamoxifen induced substantial recombination in reactive glia adjacent to the site of injury. These transgenic GFAP‐CreERT2 mice will allow the functional analysis of loxP‐modified genes in astroglia of the postnatal and adult brain.

A role for glia in the progression of Rett/'s syndrome

Rett’s syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by loss of function of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed in most tissues, loss of MeCP2 expression results primarily in neurological symptoms. Earlier studies suggested the idea that RTT is due exclusively to loss of MeCP2 function in neurons. Although defective neurons clearly underlie the aberrant behaviours, we and others showed recently that the loss of MECP2 from glia negatively influences neurons in a non-cell-autonomous fashion. Here we show that in globally MeCP2-deficient mice, re-expression of Mecp2 preferentially in astrocytes significantly improved locomotion and anxiety levels, restored respiratory abnormalities to a normal pattern, and greatly prolonged lifespan compared to globally null mice. Furthermore, restoration of MeCP2 in the mutant astrocytes exerted a non-cell-autonomous positive effect on mutant neurons in vivo, restoring normal dendritic morphology and increasing levels of the excitatory glutamate transporter VGLUT1. Our study shows that glia, like neurons, are integral components of the neuropathology of RTT, and supports the targeting of glia as a strategy for improving the associated symptoms.

Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice.

Reef coral fluorescent proteins (RCFPs) are bright fluorescent proteins (FPs) covering a wide spectral range. We used various RCFP genes to transgenically color different cell populations in the brain. The mouse Thy1.2 promoter was used to target expression of HcRed1 in neurons, the human glial fibrillary acidic protein (GFAP) promoter to label astrocytes with AmCyan1, AsRed2 and mRFP1 as well as the mouse proteolipid protein promoter to mark oligodendrocytes with DsRed1. In brain sections of transgenic mice, RCFP expression was found to be highly specific using immunohistochemistry and fluorescence microscopy. In contrast to transgenic mice with expression of jellyfish FP variants, RCFPs formed numerous fluorescent precipitates. These aggregates were primarily found in cell somata and also in cell processes. Older mice were more affected than younger ones. Despite these fluorescent deposits, physiological properties of RCFP expressing brain cells such as whole-cell membrane currents or glutamate-evoked calcium signaling seemed to be unaffected. While brightness and spectral variation of RCFPs are optimal for expression in transgenic animals used in physiological experiments, the formation of fluorescent precipitates in various cell types limits their use for morphological cell analysis in situ.

Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.

Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina.

Reactive glial cells: increased stiffness correlates with increased intermediate filament expression

Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia‐reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild‐type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP−/−Vim−/−) in which glial cells are consequently devoid of IFs, and normal Long‐Evans rats. In response to ischemia‐reperfusion, glial cells stiffened significantly in wild‐type mice and rats but were unchanged in GFAP−/− Vim−/− mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up‐regulation in reactive glial cells may facilitate neuroregeneration.—Lu, Y.‐B., Iandiev, I., Hollborn, M., Korber, N., Ulbricht, E., Hirrlinger, P. G., Pannicke, T., Wei, E.‐Q., Bringmann, A., Wol‐burg, H., Wilhelmsson, U., Pekny, M., Wiedemann, P., Reichenbach, A., Kas, J. A. Reactive glial cells: increased stiffness correlates with increased intermediate filament expression. FASEB J. 25, 624–631 (2011). www.fasebj.org

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting.

Osmotic swelling characteristics of glial cells in the murine hippocampus, cerebellum, and retina in situ

Glial cells are proposed to play a major role in the ionic and osmotic homeostasis in the CNS. Swelling of glial cells contributes to the development of edema in neural tissue under pathological conditions such as trauma and ischemia. In this study, we compared the osmotic swelling characteristics of murine hippocampal astrocytes, cerebellar Bergmann glial cells, and retinal Müller glial cells in acutely isolated tissue slices in response to hypoosmotic stress and pharmacological blockade of Kir channels. Hypoosmotic challenge induced an immediate swelling of somata in the majority of Bergmann glial cells and hippocampal astrocytes investigated, whereas Müller cell bodies displayed a substantial delay in the onset of swelling and hippocampal astroglial processes remained unaffected. Blockade of Kir channels under isoosmotic conditions had no swelling‐inducing effect in Müller cell somata but caused a swelling in brain astrocytic somata and processes. Blockade of Kir channels under hypoosmotic conditions induced an immediate and strong swelling in Müller cell somata, but had no cumulative effect to brain astroglial somata. No regulatory volume decrease could be observed in all cell types. The data suggest that Kir channels are differently implicated in cell volume homeostasis of retinal Müller cells and brain astrocytes and that Müller cells and brain astrocytes differ in their osmotic swelling properties.

Alterations in protein expression and membrane properties during Müller cell gliosis in a murine model of transient retinal ischemia

Retinal Müller glial cells are involved in K+ ion homeostasis of the tissue. Inwardly rectifying K(+) (Kir) channels play a decisive role in the process of spatial K+ buffering. It has been demonstrated that Kir-mediated currents of Müller cells are downregulated in various cases of retinal neurodegeneration. However, this has not yet been verified for any murine animal model. The aim of the present study was to investigate Müller cells after transient retinal ischemia in mice. High intraocular pressure was applied for 1h; the retina was analysed 1 week later. We studied protein expression in the tissue by immunohistochemistry, and membrane currents of isolated cells by patch-clamp experiments. We found the typical indicators of reactive gliosis such as upregulation of glial fibrillary acidic protein. Moreover, the membrane capacitance of isolated Müller cells was increased and the amplitudes of Kir-mediated currents were slightly, but significantly decreased. This murine high intraocular pressure model of transient retinal ischemia is proposed as a versatile tool for further studies on Müller cell functions in retinal degeneration.

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.

Detecting fluorescent protein expression and co-localisation on single secretory vesicles with linear spectral unmixing

Many questions in cell biology and biophysics involve the quantitation of co-localisation and the interaction of proteins tagged with different fluorophores. However, the incomplete separation of the different colour channels due to the presence of autofluorescence, along with cross-excitation and emission “bleed-through” of one colour channel into the other, all combine to render the interpretation of multi-band images ambiguous. Here we introduce a new live-cell epifluorescence spectral imaging and linear unmixing technique for classifying resolution-limited point objects containing multiple fluorophores. We demonstrate the performance of our technique by detecting, at the single-vesicle level, the co-expression of the vesicle-associated membrane protein, VAMP-2 (also called synaptobrevin-2), linked to either enhanced green fluorescent protein (EGFP) or citrine [a less pH-sensitive variant of enhanced yellow fluorescent protein (EYFP)], in mouse cortical astrocytes. In contrast, the co-expression of VAMP-2-citrine and the lysosomal transporter sialine fused to EGFP resulted in little overlap. Spectral imaging and linear unmixing permit us to fingerprint the expression of spectrally overlapping fluorescent proteins on single secretory organelles in the presence of a spectrally broad autofluorescence. Our technique provides a robust alternative to error-prone dual- or triple colour co-localisation studies.