Birgit Ledermann

General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit.

General anesthetics are widely used in clinical practice. On the molecular level, these compounds have been shown to modulate the activity of various neuronal ion channels. However, the functional relevance of identified sites in mediating essential components of the general anesthetic state, such as immobility and hypnosis, is still unknown. Using gene‐targeting technology, we generated mice harboring a subtle point mutation (N265M) in the second transmembrane region of the β3 subunit of the GABAA receptor. In these mice, the suppression of noxious‐evoked movements in response to the intravenous anesthetics etomidate and propofol is completely abolished, while only slightly decreased with the volatile anesthetics enflurane and halothane. β3(N265M) mice also display a profound reduction in the loss of righting reflex duration in response to intravenous but not volatile anesthetics. In addition, electrophysiological recordings revealed that anesthetic agents were significantly less effective in enhancing GABAA receptor‐mediated currents, and in decreasing spontaneous action potential firing in cortical brain slices derived from mutant mice. Taken together, our results demonstrate that a single molecular target, and indeed a specific residue (N265) located within the GABAA receptor β3 subunit, is a major determinant of behavioral responses evoked by the intravenous anesthetics etomidate and propofol, whereas volatile anesthetics appear to act via a broader spectrum of molecular targets.

Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury.

To investigate the role of the myelin-associated protein Nogo-A on axon sprouting and regeneration in the adult central nervous system (CNS), we generated Nogo-A-deficient mice. Nogo-A knockout (KO) mice were viable, fertile, and not obviously afflicted by major developmental or neurological disturbances. The shorter splice form Nogo-B was strongly upregulated in the CNS. The inhibitory effect of spinal cord extract for growing neurites was decreased in the KO mice. Two weeks following adult dorsal hemisection of the thoracic spinal cord, Nogo-A KO mice displayed more corticospinal tract (CST) fibers growing toward and into the lesion compared to their wild-type littermates. CST fibers caudal to the lesion-regenerating and/or sprouting from spared intact fibers-were also found to be more frequent in Nogo-A-deficient animals.

Establishment of a germ-line competent C57BL/6 embryonic stem cell line.

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.

Absence of the prion protein homologue Doppel causes male sterility

The agent that causes prion diseases is thought to be identical with PrPSc, a conformer of the normal prion protein PrPC. PrPC‐deficient mice do not exhibit major pathologies, perhaps because they express a protein termed Dpl, which shares significant biochemical and structural homology with PrPC. To investigate the physiological function of Dpl, we generated mice harbouring a homozygous disruption of the Prnd gene that encodes Dpl. Dpl deficiency did not interfere with embryonic and postnatal development, but resulted in male sterility. Dpl protein was expressed at late stages of spermiogenesis, and spermatids of Dpl mutants were reduced in numbers, immobile, malformed and unable to fertilize oocytes in vitro. Mechanical dissection of the zona pellucida partially restored in vitro fertilization. We conclude that Dpl regulates male fertility by controlling several aspects of male gametogenesis and sperm–egg interaction.

Efficient targeting of the IL-4 gene in a BALB/c embryonic stem cell line

Embryonic stem (ES) cell lines have been derived from the inner cell mass of day 3.5 blastocysts of the inbred mouse strain BALB/cJ. Twenty-three lines were karyotyped and three were selected for injection into C57BL/6J host blastocysts. Two of the three lines, BALB/c-I and BALB/c-IV, produced germ-line chimaeras. The suitability of the BALB/c-I line for gene targeting experiments was tested by transfecting a targeting construct for the interleukin-4 (IL-4) gene. Transfected BALB/c-I cells exhibited efficient homologous recombination of the targeting vector and transmitted the induced mutation through the germline. This newly-characterized BALB/c-ES cell line thus provides an alternative to the traditional 129-derived and the recently described C57BL/6 embryonic stem cell lines, and will be useful in disrupting genes involved in the immune system. Furthermore, the genetically pure BALB/c IL-4 deficient mice will aid in studying the role of IL-4 in several infectious disease models in which the BALB/c mouse is a susceptible strain.

IgD can largely substitute for loss of IgM function in B cells

The μ and δ heavy chains of IgM and IgD, the first antibody isotypes expressed during bone-marrow B-cell development, are encoded by a common transcription unit. Expression of the μ chain on the surface of late pre-B cells allows their further development to immature B cells. Coexpression of the δ chain and emigration of the immature B cells to the periphery eventually leads to the development of naive mature IgM/IgD double-positive cells. Although IgM is important in driving B-cell development, the contribution of IgD is not clear. Here we investigate the function of IgD. We generated mice deficient in IgM (IgM−/− mice) by deleting the μ region in embryonic stem cells. IgM−/− mice showed normal B-cell development and maturation, with IgD replacing membrane-bound and secretory IgM. Moreover, specific B-cell responses and isotype class switches occurred during immunization or infection. In contrast to mice deficient in B cells, IgM−/− mice survived infection with vesicular stomatitis virus by developing neutralizing immunoglobulins, but they were more susceptible than wild-type controls with delayed specific immunoglobulin responses. These data lead us to conclude that IgD is largely able to substitute for IgM functions.

Wnt signalling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression.

Wnt signalling plays an important role in both embryonic development and in tumourigenesis. Activation of the signalling cascade by wnt, but also mutations of the adenomatous polyposis coli (APC) protein and of the phosphorylation domain of beta-catenin, result in accumulation of active beta-catenin in the nucleus, where it binds to TCF/LEF transcription factors. We studied the effect of wnt signalling in embryonic stem cells by either inactivating APC or by introducing a dominant active form of beta-catenin. Both resulted in inhibition of neural differentiation in vitro and after brain grafting and in activation of downstream targets of wnt signalling, such as cyclins, c-myc, and bone morphogenetic proteins (BMP). Neural differentiation could be partially restored by the addition of the BMP antagonist noggin. This suggests a mechanism regulating the fate of differentiating embryonic stem cells.

Lymphocyte-mediated Cytotoxicity in vitro and in vivo: Mechanisms and Significance

Two classes of lymphocytes are able to exert contact-dependent cytotoxicity: cytotoxic T cells (CTL) react specifically against target cells presenting processed antigenic peptides on self MHC molecules whereas NK cells lyse a variety of target cells without classical restriction by MHC molecules. Although these two effector populations differ in specificity and lineage, the characteristics of their cytotoxic activity and some of their morphological aspects are remarkably similar (Henkart 1985). Because cytotoxicity is thought to be involved in a wide range of immune processes, the mechanisms accounting for lymphocyte-mediated cytotoxicity were studied intensively ever since cell-mediated cytolysis was first described (Govaertz 1960, Brunner et al. 1970). Important steps in the characterisation of cytolytic effector mechanisms were microscopic and microcinematographic studies showing that cytotoxic T cells form reversible conjugates with their target cells and secrete the content of their cytoplasmic granules during this process (Bykovskaja et al. 1978a,b, Matter 1979, Sanderson & Glauert 1979, Granger & Kolb 1968, Thiernesse et al. 1977, Zagury 1982), the isolation of perforin from granules of cytolytic lymphocytes and the demonstration that isolated perforin possess cytolytic activity in vitro (Podack et al. 1985. Young et al. I986a.b.c)., the observation of circular lesions on target cells lysed by lymphocytes (Dourmashkin et al. 1980, Dennert & Podack 1983) and the subsequent cloning of perforin (Shinkai et al. 1988, Lichtenheld et al. 1988) and other granule-associated proteins (Gershenfeld & Weissman 1986, Bleackley et al. 1988, Masson and

Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation

The synaptic serine protease neurotrypsin is thought to be important for adaptive synaptic processes required for cognitive functions, because humans deficient in neurotrypsin suffer from severe mental retardation. In the present study, we describe the biochemical characterization of neurotrypsin and its so far unique substrate agrin. In cell culture experiment as well as in neurotrypsin‐deficient mice, we showed that agrin cleavage depends on neurotrypsin and occurs at two conserved sites. Neurotrypsin and agrin were expressed recombinantly, purified, and assayed in vitro. A catalytic efficiency of 1.3 × 104 M−1 • s−1 was determined. Neurotrypsin activity was shown to depend on calcium with an optimal activity in the pH range of 7–8.5. Mutagenesis analysis of the amino acids flanking the scissile bonds showed that cleavage is highly specific due to the unique substrate recognition pocket of neurotrypsin at the active site. The C‐termi‐nal agrin fragment released after cleavage has recently been identified as an inactivating ligand of the Na+/ K+‐ATPase at CNS synapses, and its binding has been demonstrated to regulate presynaptic excitability. Therefore, dysregulation of agrin processing is a good candidate for a pathogenetic mechanism underlying mental retardation. In turn, these results may also shed light on mechanisms involved in cognitive functions.—Reif, R., Sales, S., Hettwer, S., Dreier, B., Gisler, C., Wolfel, J., Luscher, D., Zurlinden, A., Stephan, A., Ahmed, S., Baici, A., Ledermann, B., Kunz, B., Sonderegger, P. Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation. FASEB J. 21, 3468–3478 (2007)

Embryonic Stem Cells and Gene Targeting

The development of gene targeting technology, the exchange of an endogenous allele of a target gene for a mutated copy via homologous recombination, and the application of this technique to murine embryonic stem cells has made it possible to alter the germ‐line of mice in a predetermined way. Gene targeting has enabled researchers to generate mouse strains with defined mutations in their genome allowing the analysis of gene function in vivo. This review presents the essential tools and methodologies used for gene targeting that have been developed over the past decade. Special emphasis has been laid on the available embryonic stem cell lines and the importance of the genetic background. Also, the state‐of‐the art of gene targeting approaches in species other than mice will be discussed.