Magnetic resonance imaging and ultrasound elastography in the context of preclinical pharmacological research: significance for the 3R principles.

The 3Rs principles-reduction, refinement, replacement-are at the core of preclinical research within drug discovery, which still relies to a great extent on the availability of models of disease in animals. Minimizing their distress, reducing their number as well as searching for means to replace them in experimental studies are constant objectives in this area. Due to its non-invasive character in vivo imaging supports these efforts by enabling repeated longitudinal assessments in each animal which serves as its own control, thereby enabling to reduce considerably the animal utilization in the experiments. The repetitive monitoring of pathology progression and the effects of therapy becomes feasible by assessment of quantitative biomarkers. Moreover, imaging has translational prospects by facilitating the comparison of studies performed in small rodents and humans. Also, learnings from the clinic may be potentially back-translated to preclinical settings and therefore contribute to refining animal investigations. By concentrating on activities around the application of magnetic resonance imaging (MRI) and ultrasound elastography to small rodent models of disease, we aim to illustrate how in vivo imaging contributes primarily to reduction and refinement in the context of pharmacological research.

Mildly compromised tetrahydrobiopterin cofactor biosynthesis due to Pts variants leads to unusual body fat distribution and abdominal obesity in mice.

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.

Alternative NF-κB signaling regulates mTEC differentiation from podoplanin-expressing precursors in the cortico-medullary junction.

The thymic epithelium forms specialized niches to enable thymocyte differentiation. While the common epithelial progenitor of medullary and cortical thymic epithelial cells (mTECs and cTECs) is well defined, early stages of mTEC lineage specification have remained elusive. Here, we utilized in vivo targeting of mTECs to resolve their differentiation pathways and to determine whether mTEC progenitors participate in thymocyte education. We found that mTECs descend from a lineage committed, podoplanin (PDPN)-expressing progenitor located at the cortico-medullary junction. PDPN(+) junctional TECs (jTECs) represent a distinct TEC population that builds the thymic medulla, but only partially supports negative selection and thymocyte differentiation. Moreover, conditional gene targeting revealed that abrogation of alternative NF-κB pathway signaling in the jTEC stage completely blocked mTEC development. Taken together, this study identifies jTECs as lineage-committed mTEC progenitors and shows that NF-κB-dependent progression of jTECs to mTECs is critical to secure central tolerance.

Mice lacking phosphatase PP2A subunit PR61/B'delta (Ppp2r5d) develop spatially restricted tauopathy by deregulation of CDK5 and GSK3beta.

Functional diversity of protein phosphatase 2A (PP2A) enzymes mainly results from their association with distinct regulatory subunits. To analyze the functions of one such holoenzyme in vivo, we generated mice lacking PR61/B'δ (B56δ), a subunit highly expressed in neural tissues. In PR61/B'δ-null mice the microtubule-associated protein tau becomes progressively phosphorylated at pathological epitopes in restricted brain areas, with marked immunoreactivity for the misfolded MC1-conformation but without neurofibrillary tangle formation. Behavioral tests indicated impaired sensorimotor but normal cognitive functions. These phenotypical characteristics were further underscored in PR61/B'δ-null mice mildly overexpressing human tau. PR61/B'δ-containing PP2A (PP2A(T61δ)) poorly dephosphorylates tau in vitro, arguing against a direct dephosphorylation defect. Rather, the activity of glycogen synthase kinase-3ß, a major tau kinase, was found increased, with decreased phosphorylation of Ser-9, a putative cyclin-dependent kinase 5 (CDK5) target. Accordingly, CDK5 activity is decreased, and its cellular activator p35, strikingly absent in the affected brain areas. As opposed to tau, p35 is an excellent PP2A(T61δ) substrate. Our data imply a nonredundant function for PR61/B'δ in phospho-tau homeostasis via an unexpected spatially restricted mechanism preventing p35 hyperphosphorylation and its subsequent degradation.

Specific proteolytic cleavage of agrin regulates maturation of the neuromuscular junction.

During the initial stage of neuromuscular junction (NMJ) formation, nerve-derived agrin cooperates with muscle-autonomous mechanisms in the organization and stabilization of a plaque-like postsynaptic specialization at the site of nerve-muscle contact. Subsequent NMJ maturation to the characteristic pretzel-like appearance requires extensive structural reorganization. We found that the progress of plaque-to-pretzel maturation is regulated by agrin. Excessive cleavage of agrin via transgenic overexpression of an agrin-cleaving protease, neurotrypsin, in motoneurons resulted in excessive reorganizational activity of the NMJs, leading to rapid dispersal of the synaptic specialization. By contrast, expression of cleavage-resistant agrin in motoneurons slowed down NMJ remodeling and delayed NMJ maturation. Neurotrypsin, which is the sole agrin-cleaving protease in the CNS, was excluded as the physiological agrin-cleaving protease at the NMJ, because NMJ maturation was normal in neurotrypsin-deficient mice. Together, our analyses characterize agrin cleavage at its proteolytic α- and ß-sites by an as-yet-unspecified protease as a regulatory access for relieving the agrin-dependent constraint on endplate reorganization during NMJ maturation.

Evaluation of in vitro cultured rat oocytes, from different strains, by spindle morphology and maturation-promoting-factor activity combined with nuclear-transfer experiments.

Although successful nuclear transfer (NT) has been reported in the rat 6 years ago, somatic cell nuclear transfer (SCNT) in the rat could not be repeated. Our experiments with rat SCNT reveal the difficulties related to rat cloning. We first focussed on the most appropriate rat strain that could be used as an oocyte donor. Then we describe how rat oocytes can be kept in a nonactivated state during in vitro culture, because the latter undergo spontaneous partial activation through rapid extrusion of the second polar body after isolation from the oviduct. In the SCNT experiments performed with the one-step manipulation technique it was possible to produce rat embryos, which developed in vivo up to the blastocyst stage. In addition, we identified the implantation sites of SCNT rat embryos reconstructed with Sprague-Dawley (SD) oocytes. Furthermore, different rat strains were used as oocyte donors and their oocytes were cultured under different conditions to establish a stable nonactivating oocyte culture system. The ratio of activated to nonactivated oocytes was measured by spindle-stability and maturation promoting factor (MPF) activity. These measurements indicated that a substrain of the SD rat strain, the so-called OFA-SD strain, is the one providing the most stable oocytes, when their oocytes are cultured in the presence of the proteasome inhibitor MG132. However, it was not possible to obtain any implantation sites with reconstructed oocytes derived from the OFA-SD strain transferred to foster mothers. This goal was not achieved, even when the trichostatin A (TSA) treatment was used, which is known to enhance the cloning efficiency of reconstructed mouse, porcine, bovine, and rabbit oocytes both in vitro and in vivo by enhancing the reprogramming efficiency of the recipient nucleus.

Expression profiling in transgenic FVB/N embryonic stem cells overexpressing STAT3.

BACKGROUND: The transcription factor STAT3 is a downstream target of the LIF signalling cascade. LIF signalling or activation is sufficient to maintain embryonic stem (ES) cells in an undifferentiated and pluripotent state. To further investigate the importance of STAT3 in the establishment of ES cells we have in a first step derived stable pluripotent embryonic stem cells from transgenic FVB mice expressing a conditional tamoxifen dependent STAT3-MER fusion protein. In a second step, STAT3-MER overexpressing cells were used to identify STAT3 pathway-related genes by expression profiling in order to identify new key-players involved in maintenance of pluripotency in ES cells. RESULTS: Transgenic STAT3-MER blastocysts yielded pluripotent germline-competent ES cells at a high frequency in the absence of LIF when established in tamoxifen-containing medium. Expression profiling of tamoxifen-induced transgenic FVB ES cell lines revealed a set of 26 genes that were markedly up- or down-regulated when compared with wild type cells. The expression of four of the up-regulated genes (Hexokinase II, Lefty2, Pramel7, PP1rs15B) was shown to be restricted to the inner cell mass (ICM) of the blastocysts. These differentially expressed genes represent potential candidates for the maintenance of pluripotency of ES cells. We finally overexpressed two candidate genes, Pem/Rhox5 and Pramel7, in ES cells and demonstrated that their overexpression is sufficient for the maintenance of expression of ES cell markers as well as of the typical morphology of pluripotent ES cells in absence of LIF. CONCLUSION: Overexpression of STAT3-MER in the inner cell mass of blastocyst facilitates the establishment of ES cells and induces the upregulation of potential candidate genes involved in the maintenance of pluripotency. Two of them, Pem/Rhox5 and Pramel7, when overexpressed in ES cells are able to maintain the embryonic stem cells in a pluripotent state in a LIF independent manner as STAT3 or Nanog.

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 x 10(4) M(-1) x 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-terminal 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.

Strong and ubiquitous expression of transgenes targeted into the beta-actin locus by Cre/lox cassette replacement.

Conventional approaches to produce transgenic mice recurrently yield unpredictable patterns and levels of transgene expression, a situation calling for the development of new techniques to overcome these drawbacks in the context of overexpression studies. Here we present an efficient method for rapid and reproducible transgenesis using the recombinase mediated cassette exchange (RMCE) (Bouhassira et al.: Blood 90:3332-3344, 1997) procedure. A lox511-EGFP-TK/neo-loxP cassette was placed under the control of the endogenous mouse beta-actin promoter. Heterozygous mice revealed strong and ubiquitous EGFP expression throughout embryogenesis and adulthood. Reproducibly, the same expression pattern was obtained with RMCE when it was used to replace the EGFP-harboring cassette by ECFP or placental alkaline phosphatase (PLAP) reporter genes (DePrimo et al.: Transgenic Res 5:459-466, 1996). Furthermore, the RMCE procedure proved efficient as well in embryonic stem (ES) cells as directly in zygotes. Our results demonstrate ubiquitous expression of floxed transgenes in the endogenous beta-actin locus and they support the general use of the beta-actin locus for targeted transgenesis.

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.

Antiviral immune responses in gene-targeted mice expressing the immunoglobulin heavy chain of virus-neutralizing antibodies.

Two gene-targeted immunoglobulin heavy chain transgenic mouse strains, TgH(KL25) and TgH(VI10), expressing neutralizing specificities for lymphocytic choriomeningitis virus and vesicular stomatitis virus, respectively, have been generated. Three days after lymphocytic choriomeningitis virus infection, TgH(KL25) mice showed a thymus-independent neutralizing IgM response followed by thymus-dependent (TD) IgG. In contrast, WT mice mounted only a TD IgG response around day 80. These observations indicated that not only structural properties of the virus but also immunological parameters such as the frequency of B cells were indicative for the induction of thymus-independent versus TD Ig responses. Naïve vesicular stomatitis virusspecific Ig heavy chain transgenic mice displayed greatly elevated natural antibody titers. However, despite these high naïve titers, de novo activation of naïve CD4+ T and B cells was not blocked. Therefore, B cells giving rise to natural antibodies do not participate in virus-induced antibody responses.

Targeted disruption of the mouse PAS domain serine/threonine kinase PASKIN.

PASKIN is a novel mammalian serine/threonine kinase containing two PAS (Per-Arnt-Sim) domains. PASKIN is related to the Rhizobium oxygen sensor protein FixL and to AMP-regulated kinases. Like FixL, the sensory PAS domain of PASKIN controls the kinase activity by autophosphorylation in a (unknown) ligand-dependent manner. In Saccharomyces cerevisiae, the two PASKIN orthologues PSK1 and PSK2 phosphorylate three translation factors and two enzymes involved in glycogen synthesis, thereby coordinately regulating protein synthesis and glycolytic flux. To elucidate the function of mammalian PASKIN, we inactivated the mouse Paskin gene by homologous recombination in embryonic stem cells. Paskin(-/-) mice showed normal development, growth, and reproduction. The targeted integration of a lacZ reporter gene allowed the identification of the cell types expressing mouse PASKIN. Surprisingly, PASKIN expression is strongly upregulated in postmeiotic germ cells during spermatogenesis. However, fertility and sperm production and motility were not affected by the PASKIN knockout. The Ppp1r7 gene encoding Sds22, a regulatory subunit of protein phosphatase 1, shares the promoter region with the Paskin gene, pointing towards a common transcriptional regulation. Indeed, Sds22 colocalized with the cell types expressing PASKIN in vivo, suggesting a functional role of protein phosphatase-1 in the regulation of PASKIN autophosphorylation.

Dwarfism and low insulin-like growth factor-1 due to dopamine depletion in Pts-/- mice rescued by feeding neurotransmitter precursors and H4-biopterin.

The tetrahydrobiopterin (BH4) cofactor is essential for the biosynthesis of catecholamines and serotonin and for nitric-oxide synthase (NOS). Alterations in BH4 metabolism are observed in various neurological and psychiatric diseases, and mutations in one of the human metabolic genes causes hyperphenylalaninemia and/or monoamine neurotransmitter deficiency. We report on a knockout mouse for the Pts gene, which codes for a BH4-biosynthetic enzyme. Homozygous Pts-/- mice developed with normal morphology but died after birth. Upon daily oral administration of BH4 and neurotransmitter precursors the Pts-/- mice eventually survived. However, at sexual maturity (6 weeks) the mice had only one-third of the normal body weight and were sexually immature. Biochemical analysis revealed no hyperphenylalaninemia, normal brain NOS activity, and almost normal serotonin levels, but brain dopamine was 3% of normal. Low dopamine leads to impaired food consumption as reflected by the severe growth deficiency and a 7-fold reduced serum insulin-like growth factor-1 (IGF-1). This is the first link shown between 6-pyruvoyltetrahydropterin synthase- or BH4-biosynthetic activity and IGF-1.

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.

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 beta3 subunit of the GABA(A) 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. beta3(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 GABA(A) 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 GABA(A) receptor beta3 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.

Absence of the prion protein homologue Doppel causes male sterility.

The agent that causes prion diseases is thought to be identical with PrP(Sc), a conformer of the normal prion protein PrP(C). PrP(C)-deficient mice do not exhibit major pathologies, perhaps because they express a protein termed Dpl, which shares significant biochemical and structural homology with PrP(C). 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.

Fliih, a gelsolin-related cytoskeletal regulator essential for early mammalian embryonic development.

The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation.