IsoQC (QPCTL) knock-out mice suggest differential substrate conversion by glutaminyl cyclase isoenzymes.

Secretory peptides and proteins are frequently modified by pyroglutamic acid (pE, pGlu) at their N-terminus. This modification is catalyzed by the glutaminyl cyclases QC and isoQC. Here, we decipher the roles of the isoenzymes by characterization of IsoQC-/- mice. These mice show a significant reduction of glutaminyl cyclase activity in brain and peripheral tissue, suggesting ubiquitous expression of the isoQC enzyme. An assay of substrate conversion in vivo reveals impaired generation of the pGlu-modified C-C chemokine ligand 2 (CCL2, MCP-1) in isoQC-/- mice. The pGlu-formation was also impaired in primary neurons, which express significant levels of QC. Interestingly, however, the formation of the neuropeptide hormone thyrotropin-releasing hormone (TRH), assessed by immunohistochemistry and hormonal analysis of hypothalamic-pituitary-thyroid axis, was not affected in isoQC-/-, which contrasts to QC-/-. Thus, the results reveal differential functions of isoQC and QC in the formation of the pGlu-peptides CCL2 and TRH. Substrates requiring extensive prohormone processing in secretory granules, such as TRH, are primarily converted by QC. In contrast, protein substrates such as CCL2 appear to be primarily converted by isoQC. The results provide a new example, how subtle differences in subcellular localization of enzymes and substrate precursor maturation might influence pGlu-product formation.

Screen for alterations of iron related parameters in N-ethyl-N-nitrosourea-treated mice identified mutant lines with increased plasma ferritin levels.

Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.

Glutaminyl cyclase-mediated toxicity of pyroglutamate-beta amyloid induces striatal neurodegeneration.

BACKGROUND: Posttranslational modifications of beta amyloid (Aß) have been shown to affect its biophysical and neurophysiological properties. One of these modifications is N-terminal pyroglutamate (pE) formation. Enzymatic glutaminyl cyclase (QC) activity catalyzes cyclization of truncated Aß(3-x), generating pE3-Aß. Compared to unmodified Aß, pE3-Aß is more hydrophobic and neurotoxic. In addition, it accelerates aggregation of other Aß species. To directly investigate pE3-Aß formation and toxicity in vivo, transgenic (tg) ETNA (E at the truncated N-terminus of Aß) mice expressing truncated human Aß(3-42) were generated and comprehensively characterized. To further investigate the role of QC in pE3-Aß formation in vivo, ETNA mice were intercrossed with tg mice overexpressing human QC (hQC) to generate double tg ETNA-hQC mice. RESULTS: Expression of truncated Aß(3-42) was detected mainly in the lateral striatum of ETNA mice, leading to progressive accumulation of pE3-Aß. This ultimately resulted in astrocytosis, loss of DARPP-32 immunoreactivity, and neuronal loss at the sites of pE3-Aß formation. Neuropathology in ETNA mice was associated with behavioral alterations. In particular, hyperactivity and impaired acoustic sensorimotor gating were detected. Double tg ETNA-hQC mice showed similar Aß levels and expression sites, while pE3-Aß were significantly increased, entailing increased astrocytosis and neuronal loss. CONCLUSIONS: ETNA and ETNA-hQC mice represent novel mouse models for QC-mediated toxicity of truncated and pE-modified Aß. Due to their significant striatal neurodegeneration these mice can also be used for analysis of striatal regulation of basal locomotor activity and sensorimotor gating, and possibly for DARPP-32-dependent neurophysiology and neuropathology. The spatio-temporal correlation of pE3-Aß and neuropathology strongly argues for an important role of this Aß species in neurodegenerative processes in these models.

Dual function of the UNC-45b chaperone with myosin and GATA4 in cardiac development.

Cardiac development requires interplay between the regulation of gene expression and the assembly of functional sarcomeric proteins. We report that UNC-45b recessive loss-of-function mutations in C3H and C57BL/6 inbred mouse strains cause arrest of cardiac morphogenesis at the formation of right heart structures and failure of contractile function. Wild-type C3H and C57BL/6 embryos at the same stage, E9.5, form actively contracting right and left atria and ventricles. The known interactions of UNC-45b as a molecular chaperone are consistent with diminished accumulation of the sarcomeric myosins, but not their mRNAs, and the resulting decreased contraction of homozygous mutant embryonic hearts. The novel finding that GATA4 accumulation is similarly decreased at the protein but not mRNA levels is also consistent with the function of UNC-45b as a chaperone. The mRNAs of known downstream targets of GATA4 during secondary cardiac field development, the cardiogenic factors Hand1, Hand2 and Nkx-2.5, are also decreased, consistent with the reduced GATA4 protein accumulation. Direct binding studies show that the UNC-45b chaperone forms physical complexes with both the alpha and beta cardiac myosins and the cardiogenic transcription factor GATA4. Co-expression of UNC-45b with GATA4 led to enhanced transcription from GATA promoters in naïve cells. These novel results suggest that the heart-specific UNC-45b isoform functions as a molecular chaperone mediating contractile function of the sarcomere and gene expression in cardiac development.

Selective hippocampal neurodegeneration in transgenic mice expressing small amounts of truncated Aß is induced by pyroglutamate-Aß formation.

Posttranslational amyloid-ß (Aß) modification is considered to play an important role in Alzheimer's disease (AD) etiology. An N-terminally modified Aß species, pyroglutamate-amyloid-ß (pE3-Aß), has been described as a major constituent of Aß deposits specific to human AD but absent in normal aging. Formed via cyclization of truncated Aß species by glutaminyl cyclase (QC; QPCT) and/or its isoenzyme (isoQC; QPCTL), pE3-Aß aggregates rapidly and is known to seed additional Aß aggregation. To directly investigate pE3-Aß toxicity in vivo, we generated and characterized transgenic TBA2.1 and TBA2.2 mice, which express truncated mutant human Aß. Along with a rapidly developing behavioral phenotype, these mice showed progressively accumulating Aß and pE3-Aß deposits in brain regions of neuronal loss, impaired long-term potentiation, microglial activation, and astrocytosis. Illustrating a threshold for pE3-Aß neurotoxicity, this phenotype was not found in heterozygous animals but in homozygous TBA2.1 or double-heterozygous TBA2.1/2.2 animals only. A significant amount of pE3-Aß formation was shown to be QC-dependent, because crossbreeding of TBA2.1 with QC knock-out, but not isoQC knock-out, mice significantly reduced pE3-Aß levels. Hence, lowering the rate of QC-dependent posttranslational pE3-Aß formation can, in turn, lower the amount of neurotoxic Aß species in AD.

The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions.

Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE(1) -CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis.

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters.

Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.

Glutaminyl cyclase knock-out mice exhibit slight hypothyroidism but no hypogonadism: implications for enzyme function and drug development.

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development.

Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo.

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability in vivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in plasma enzyme activities as novel organ-specific disease models.

Measurement of plasma enzyme activities is part of routine medical examination protocols and provides valuable parameters for the diagnosis of various organ diseases. In the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project, clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma enzyme activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, alpha-amylase and creatine kinase. We identified a large number of animals that consistently exhibited altered plasma enzyme activities. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for each parameter. Breeding experiments in selected lines detected the linkage of the causative mutations to defined chromosomal regions. Subsequently, identification of the mutated genes was successfully carried out in chosen lines, resulting in a novel alkaline phosphatase liver/bone/kidney (Alpl) alteration in one line and the strong indication for a dystrophin (Dmd) alteration in another line. The mouse mutants with abnormal plasma enzyme activities recovered in the Munich ENU project are novel tools for the systematic dissection of the pathogenesis of organ diseases.

TRESK two-pore-domain K+ channels constitute a significant component of background potassium currents in murine dorsal root ganglion neurones.

TRESK (TWIK-related spinal cord K(+) channel) is the most recently identified member of the two-pore-domain potassium channel (K(2P)) family, the molecular source of background potassium currents. Human TRESK channels are not affected by external acidification. However, the mouse orthologue displays moderate pH dependence isolated to a single histidine residue adjacent to the GYG selectivity filter. In the human protein, sequence substitution of tyrosine by histidine at this critical position generated a mutant that displays almost identical proton sensitivity compared with mouse TRESK. In contrast to human TRESK, which is specifically located in spinal cord, we detected mouse TRESK (mTRESK) mRNA in several epithelial and neuronal tissues including lung, liver, kidney, brain and spinal cord. As revealed by endpoint and quantitative RT-PCR, mTRESK channels are mainly expressed in dorsal root ganglia (DRG) and on the transcript level represent the most important background potassium channel in this tissue. DRG neurones of all sizes were labelled by in situ hybridizations with TRESK-specific probes. In DRG neurones of TRESK[G339R] functional knock-out (KO) mice the standing outward current IK(so) was significantly reduced compared with TRESK wild-type (WT) littermates. Different responses to K(2P) channel regulators such as bupivacaine, extracellular protons and quinidine corroborated the finding that approximately 20% of IK(so) is carried by TRESK channels. Unexpectedly, we found no difference in resting membrane potential between DRG neurones of TRESK[WT] and TRESK[G339R] functional KO mice. However, in current-clamp recordings we observed significant changes in action potential duration and amplitude of after-hyperpolarization. Most strikingly, cellular excitability of DRG neurones from functional KO mice was significantly augmented as revealed by reduced rheobase current to elicit action potentials.

Use of an exon-trapping vector for the evaluation of splice-site mutations.

Prediction of the effects of splice-site variations by sequence analysis is difficult. In this study we provide the means for a rapid evaluation of the potential for splice-site mutations to interfere with RNA processing. The system may be useful in reverse genetics or mapping studies when isolation and characterization of mRNA is arduous or not possible. In the assay we cloned wild-type and mutant sequences of murine splice-site mutations into an exon-trapping vector and characterized splicing of both recombinant transcripts in a transient cell culture system. Results from this artificial assay were compared with in vivo data from the respective mouse models. We found that the exon-trapping system allows one to confidently predict whether a splice-site variation is going to have a splicing effect in vivo, but the system does not always reflect in vivo splicing in detail. In summary, the exon-trapping system is a reliable and easy-to-use tool for a first evaluation of splice effects.

N-ethyl-N-nitrosourea-based generation of mouse models for mutant G protein-coupled receptors.

Chemical random mutagenesis techniques with the germ line supermutagen N-ethyl-N-nitrosourea (ENU) have been established to provide comprehensive collections of mouse models, which were then mined and analyzed in phenotype-driven studies. Here, we applied ENU mutagenesis in a high-throughput fashion for a gene-driven identification of new mutations. Selected members of the large superfamily of G protein-coupled receptors (GPCR), melanocortin type 3 (Mc3r) and type 4 (Mc4r) receptors, and the orphan chemoattractant receptor GPR33, were used as model targets to prove the feasibility of this approach. Parallel archives of DNA and sperm from mice mutagenized with ENU were screened for mutations in these GPCR, and in vitro assays served as a preselection step before in vitro fertilization was performed to generate the appropriate mouse model. For example, mouse models for inherited obesity were established by selecting fully or partially inactivating mutations in Mc4r. Our technology described herein has the potential to provide mouse models for a GPCR dysfunction of choice within <4 mo and can be extended to other gene classes of interest.

Current methods for high-throughput detection of novel DNA polymorphisms.

For research varying from the identification of specific disease loci to the investigation of protein function, the detection of DNA sequence variations requires reliable methods. Technologies enabling rapid and cost effective identification of novel genetic polymorphisms will significantly impact future work in genetic mapping studies, drug target discovery and validation and pharmacogenomics.:

Heterozygous R1101K mutation of the DCTN1 gene in a family with ALS and FTD.

A heterozygous R1101K mutation of the p150 subunit of dynactin (DCTN1) is reported in a family with amyotrophic lateral sclerosis (ALS) and co-occurrence of frontotemporal dementia (FTD). Two members of our kindred were affected with motor neuron disease and two with dementia in an autosomal dominant pattern of inheritance. We excluded the involvement of the ALS and FTD-linked genes for copper/zinc superoxide dismutase (SOD1) and tau. The R1101K sequence alteration of the DCTN1 gene may predispose subjects to ALS and FTD.

Negative-strand RNA viral vectors: intravenous application of Sendai virus vectors for the systemic delivery of therapeutic genes.

Treatment by gene replacement is critical in the field of gene therapy. Suitable vectors for the delivery of therapeutic genes have to be generated and tested in preclinical settings. Recently, extraordinary features for a local gene delivery by Sendai virus vectors (SeVV) have been reported for different tissues. Here we show that direct intravenous application of SeVV in mice is not only feasible and safe, but it results in the secretion of therapeutic proteins to the circulation, for example, human clotting Factor IX (hFIX). In vitro characterization of first-generation SeVV demonstrated that secreted amounts of hFIX were at least comparable to published results for retroviral or adeno-associated viral vectors. Furthermore, as a consideration for application in humans, SeVV transduction led to efficient hFIX synthesis in primary human hepatocytes, and SeVV-encoded hFIX proteins could be shown to be functionally active in the human clotting cascade. In conclusion, our investigations demonstrate for the first time that intravenous administration of negative-strand RNA viral vectors may become a useful tool for the wide area of gene replacement requirements.

Random mutagenesis in the mouse as a tool in drug discovery.

The flood of raw information generated by large-scale data acquisition technologies in genomics, microarrays and proteomics is changing the early stages of the drug discovery process. Although many more potential drug targets are now available compared with the pre-genomics era, knowledge about the physiological context in which these targets act--information crucial to both discovery and development--is scarce. Random mutagenesis strategies in the mouse provide scalable approaches for both the gene-driven validation of candidate targets in vivo and the discovery of new physiological pathways by phenotype-driven screens.