Mammalian mitochondrial nitric oxide synthase: Characterization of a novel candidate

AbstractRecently a novel family of putative nitric oxide synthases, with AtNOS1, the plant member implicated in NO production, has been described. Here we present experimental evidence that a mammalian ortholog of AtNOS1 protein functions in the cellular context of mitochondria. The expression data suggest that a candidate for mammalian mitochondrial nitric oxide synthase contributes to multiple physiological processes during embryogenesis, which may include roles in liver haematopoesis and bone development

Modelling neurofibromatosis type 1 tibial dysplasia and its treatment with lovastatin

<p>Abstract</p> <p>Background</p> <p>Bowing and/or pseudarthrosis of the tibia is a known severe complication of neurofibromatosis type 1 (NF1). Mice with conditionally inactivated neurofibromin (Nf1) in the developing limbs and cranium (Nf1Prx1) show bowing of the tibia caused by decreased bone mineralisation and increased bone vascularisation. However, in contrast to NF1 patients, spontaneous fractures do not occur in Nf1Prx1 mice probably due to the relatively low mechanical load. We studied bone healing in a cortical bone injury model in Nf1Prx1 mice as a model for NF1-associated bone disease. Taking advantage of this experimental model we explore effects of systemically applied lovastatin, a cholesterol-lowering drug, on the Nf1 deficient bone repair.</p> <p>Methods</p> <p>Cortical injury was induced bilaterally in the <it>tuberositas tibiae </it>in Nf1Prx1 mutant mice and littermate controls according to a method described previously. Paraffin as well as methacrylate sections were analysed from each animal. We divided 24 sex-matched mutant mice into a lovastatin-treated and an untreated group. The lovastatin-treated mice received 0.15 mg activated lovastatin by daily gavage. The bone repair process was analysed at three consecutive time points post injury, using histological methods, micro computed tomography measurements and <it>in situ </it>hybridisation. At each experimental time point, three lovastatin-treated mutant mice, three untreated mutant mice and three untreated control mice were analysed. The animal group humanely killed on day 14 post injury was expanded to six treated and six untreated mutant mice as well as six control mice.</p> <p>Results</p> <p>Bone injury repair is a complex process, which requires the concerted effort of numerous cell types. It is initiated by an inflammatory response, which stimulates fibroblasts from the surrounding connective tissue to proliferate and fill in the injury site with a provisional extracellular matrix. In parallel, mesenchymal progenitor cells from the periost are recruited into the injury site to become osteoblasts. In Nf1Prx1 mice bone repair is delayed and characterised by the excessive formation and the persistence of fibro-cartilaginous tissue and impaired extracellular matrix mineralisation. Correspondingly, expression of Runx2 is downregulated. High-dose systemic lovastatin treatment restores Runx2 expression and accelerates new bone formation, thus improving cortical bone repair in Nf1Prx1 tibia. The bone anabolic effects correlate with a reduction of the mitogen activated protein kinase pathway hyper-activation in Nf1-deficient cells.</p> <p>Conclusion</p> <p>Our data suggest the potential usefulness of lovastatin, a drug approved by the US Food and Drug Administration in 1987 for the treatment of hypercholesteraemia, in the treatment of Nf1-related fracture healing abnormalities. The experimental model presented here constitutes a valuable tool for the pre-clinical stage testing of candidate drugs, targeting Nf1-associated bone dysplasia.</p

Functional characterization of neurofibromin in the development of the musculoskeletal system

Neurofibromatose Typ 1 (NF1) ist eine autosomal dominant vererbte Multiorganerkrankung, die schätzungsweise bei einem Kind von 2500-3000 Geburten auftritt. Das Krankheitsbild eines NF1 Patienten ist gekennzeichnet durch Hautveränderungen, den sog. Café-au-lait-Flecken sowie subkutanen und plexiformen Neurofibromen. Zusätzliche Symptome sind Lisch-Knötchen in den Augen, mentale Retardierung und Epilepsieanfälle. Spezifische Ver-änderungen des muskuloskelettale Systems sind Kleinwuchs, Skoliose, Pseudarthrose, Osteo- porose, tibiale Dysplasie sowie eine verminderte Muskelkraft. Das krankheitsverursachende NF1 Gen lokalisiert auf Chromosom 17 und kodiert das kleine GTPase aktivierende Protein Neurofibromin, welches eine Funktion als Tumorsuppressor ausübt. Vererbte als auch de novo Mutationen im NF1 Gen führen zu einem nicht-funktionsfähigen Protein, einer Hyperaktivierung des p21 RAS Proteins und einem dysregulierten Zellteilungsverhalten durch Aktivierung der RAS/MAPK-Signalkaskade. Das Ziel der Dissertation war die funktionelle Charakterisierung von Neurofibromin in der Entwicklung des muskuloskelettalen Systems. Dafür wurde ein konditionelles Knockout Maussystems etabliert, in dem Nf1 in undifferenzierten mesenchymalen Vorläuferzellen der sich entwickelnden Extremitäten inaktiviert wurde (Nf1Prx1). Der daraus resultierende Nf1Prx1 Mausphänotyp wurde hinsichtlich seines muskuloskelettalen Phänotyps charakterisiert. Die Nf1Prx1 Mäuse waren kleiner und die Skelettelemente in ihrer Größe reduziert. Histolo-gische Untersuchungen, Genexpressionsstudien und in vivo Analysen belegten, dass in Folge der Aktivierung der RAS/MAPK- Signaltransduktion die Chondrozytenproliferation verringert war und die Chondrozytenzonen in der Wachstumsfuge verkürzt vorlagen. Das wohl auffälligste Merkmal neugeborener Nf1Prx1 Mausmutanten waren die fusionierten Hüftgelenke, wodurch die Hinterläufe hinterhergezogen wurden und eine normale Fortbewe-gung verhindert wurde. Dieser Phänotyp entstand in der pränatalen Entwicklung durch die gestörte Ausbildung eines Gelenkspalts, welcher normalerweise zwischen den Entwicklungs-stadien E13.5 und E14.5 gebildet wird. Anhand einer durchgängigen Col2a1 Genexpression und einem persistent nukleär lokalisiertem SOX9 Protein konnte gezeigt werden, dass die Zellen des Gelenkspalts weiterhin chondrogenen Charakter besaßen und demnach die Knor- pelelemente von Pelvis und Femur miteinander verschmolzen blieben. Auch andere Gelenke wie Ellenbogen- und Kniegelenk waren in der Nf1Prx1 Maus deformiert. Histologische Analy-sen prä- und postnataler Entwicklungsstadien zeigten, dass dieser Defekt auf einen zuneh-menden Degradationsprozess in der postnatalen Entwicklung zurückzuführen ist. Die chondrozytenspezifische Ablation von Nf1 in einem weiteren Mausmodell (Nf1Col2) führte hingegen nicht zu einem Gelenkphänotyp. Zusätzlich zu den Skelett-Abnormalitäten zeigten Nf1Prx1 Mutanten starke Veränderungen des Muskelapparates. Am Beispiel des M.triceps konnte demonstriert werden, dass der Muskel kleiner war, die Anzahl an Muskelfasern pro Muskel reduziert und der Anteil an Fett- und Bindegewebe im Muskel dramatisch erhöht war. Damit ging eine verringerte Zugkraft der Vorderextremitäten der Mäuse einher. Um zu prüfen, ob der Muskelphänotyp auf einen prä-natalen Entwicklungsdefekt zurückzuführen ist, wurden die einzelnen Phasen der Myogenese näher betrachtet. Immunhistochemische Analysen der frühen Myogenese demonstrierten, dass weder Migrations- noch Proliferationsprozess der Muskelvorläuferzellen in den Nf1Prx1 Mausmutanten betroffen waren. Hingegen konnte anhand der verminderten Genexpressionen von Markern der Muskeldifferenzierung wie Myogenin und MyoD ein Muskeldifferenzie-rungsdefekt in der Nf1Prx1 Mutante gezeigt werden. Zudem war die Proliferationsrate der Myoblasten erhöht und das MYOD Protein vornehmlich nukleär in den Muskelfibrillen loka-lisiert. Zusammenfassend ist zu konstatieren, dass Neurofibromin in verschiedenen Aspekten der muskuloskelettelen Entwicklung eine wichtige Rolle spielt. Die Inaktivierung von Nf1 resul-tiert in einem Muskel- und Gelenkphänotyp und läßt damit auf einen direkten Zusammenhang in der Entwicklung der einzelnen Komponenten des muskuloskelettalen Systems schliessen. Das Nf1Prx1 Mausmodell eignet sich als Modellsystem für die humane NF1 Erkrankung, da es wichtige Aspekte des humanen muskuloskelettalen NF1 Phänotyps rekapituliert und damit das Entschlüsseln des Pathomechanismus ermöglicht.Neurofibromatosis Type 1 (NF1) is a tumor predisposition syndrome. It is inherited in an au-tosomal dominant manner and affects 1 in 2500-3000 individuals. NF1 is mainly characterized by the occurrence of pigmented skin lesions (café-au-lait spots) and the formation of neurofi-bromas along central and peripheral nerves. In addition to that, patients suffer from iris Lisch nodules, mental retardation, learning disabilities and epilepsy. Furthermore, NF1 patients show musculoskeletal lesions such as short stature, scoliosis, pseudarthrosis, osteoporosis, tibial dysplasia and a diminished muscle force. The affected gene Neurofibromin 1 (NF1) is located on Chromosom 17. The protein negative-ly regulates a key signal transduction protein called p21 RAS. Thus, loss of NF1 activity is expected to lead to the inability to shut off activated p21 RAS with subsequent aberrant growth promoting signals. In this work I was able to establish a conditional inactivation approach to ablate Nf1 function specifically in the early undifferentiated cells of the developing limbs. Loss of Neurofibromin leads to a severe musculoskeletal phenotype (Nf1Prx1). Inactivation of Nf1 resulted in short stature and mice exhibited congenital limb shortening. Measurements of bone growth, histology, gene expression profiling and in vivo analysis re-vealed diminished chondrocyte proliferation and a differentiation defect due to the activation of RAS/MAPK signaling. Newborn Nf1Prx1 mice were easily recognized at birth due to their inability to bend their hind limbs at the hip joint. Examinations displayed cartilaginous fusions of the hip joints. This phenotype resulted from a lack of joint cavity initiation caused by an impaired chondrocyte de- differentiation in the presumptive joint region. This was shown by a continous expression pattern of Col2a1, a reduced Gdf5 expression and a nuclear SOX9 localization persisted in all cells of the cartilage anlage in Nf1Prx1 mice. Elbow and knee joint were severely deformed, too. Histological analysis indicated a postnatal degradation process of elbow and knee joint. In contrast to that, chondrocyte specific inactivation of Nf1 did not lead to fused and mis-shaped joints. Furthermore, loss of Nf1 resulted in a muscle phenotype. In M. triceps, for ex-ample, muscle size was reduced, number of muscle fibers diminished but connective tissue and fat was dramatically increased in the mutant muscle. Nf1Prx1 mice had also diminished grip strength. Detailed analysis of myogenesis revealed no differences in migration and early proliferation of muscle precursors in the mutant mice. In contrast, gene expression of muscle differentiation markers Myogenin and MyoD was reduced. Besides myoblast proliferation was increased and MYOD protein primarily localized in the nuclei of Desmin positive muscle fibres. These results proposed a defect in differentiation of Nf1Prx1 muscles. In fact, Nf1 has essential multiple roles in skeletal and muscle development and therefore promotes assumptions on a direct developmental relation of musculoskeletal components. Nf1Prx1 mouse model recapitulates the human musculoskeletal NF1 phenotype and can be used for understanding pathomechanism and the establishment of prospective therapy approaches

Overview of compounds used for training and evaluation of classifiers.

<p>The table lists all compounds along with their carcinogenicity class and CAS Registry Number. Corn oil (CO) or 0.1% carboxymethyl cellulose (CMC) was used as vehicle. Doses were selected for each compound based on the tumorigenic dose rate 50 (TD₅₀), long-term animal studies leading to liver cancer known from the literature or initial dose range-finding studies. After a dosing period of 3 or 14 days the mouse livers were subjected to gene expression analysis using an Affymetrix platform. Two treatment groups, each comprising 5–6 male and female mice, respectively, were examined for each compound, dose and point of time. Time matched control groups were included for both vehicles. Each treatment group has a unique group ID which is composed of the compound short name, the group number and the sex (Female or Male).</p

Performance comparison with signatures known from the literature.

<p>(<b>A</b>) The grouped bar plots depict the area under the ROC curves obtained for novel and known signatures for the separation of C and NC after 3 days of treatment. For this purpose, the performance of diverse classifiers was evaluated by a 3-fold cross-validation. Each bar corresponds to a certain classifier (see legend) and each group of bars refers to a certain signature. The horizontal dashed line indicates the performance that would have been achieved by random guessing. The adjacent Venn diagrams illustrate informative genes common between signatures. (<b>B</b>) Same plots as in (A), but for C vs. NC classification after 14 days of repeated dosing. (<b>C</b>) Mean ROC scores and signature overlaps for GC vs. NGC classification after 3 days of treatment. (<b>D</b>) Same plots as in (C), but for GC vs. NGC classification after 14 days of repeated dosing.</p

Classification results obtained for different signatures.

<p>(<b>A</b>) The four heatmaps show the predictions resulting from diverse binary classifiers for the discrimination of C from NC after 3 days (left two heatmaps) or 14 days (right two heatmaps) of repeated dosing. For each dosing time two heatmaps are depicted which correspond to male and female mice, respectively. The rows correspond to different classifiers and the columns to different treatment groups. The continuous prediction scores, returned from the classifiers, were transformed to confidence scores between 0 and 1, which provide an estimate of the probability of class C. The colorbar on top shows the true class annotation. The black vertical lines separate the test samples from the three folds of cross-validation. (<b>B</b>) Heatmaps illustrating the classification outcome of diverse predictors to distinguish GC from NGC based on characteristic gene expression profiles observed in male and female mouse liver samples after 3 or 14 days of administration. KNN, K-Nearest Neighbor; SVM, Support Vector Machine; PAM, Prediction Analysis for Microarrays.</p

Reclassification of the compounds CPA, TAA, and WY.

<p>(<b>A</b>) Points indicate treatment groups which were originally represented by a vector containing the fold-changes of all signature genes and then transformed to its two principal components. Here, the informative genes for discriminating GC vs. NGC after 3 days of repeated dosing were used. Groups of male animals are drawn as squares and female ones as circles. The fill color of the points indicates the compound class. Polygons indicate the convex hulls of clusters corresponding to either male or female mice treated with a certain class of compounds. (<b>B</b>) Similar plot as in (A), but the multi-gene signature for GC vs. NGC classification after 14 days was used here. (<b>C</b>) The heatmap provides a graphical representation of the fold-changes of 15 selected signature genes from the 14-day signature for GC vs. NGC classification. Rows correspond to genes and columns to treatment groups. Upregulated genes are colored in red and downregulated ones in green. The colorbar on top indicates the corresponding compound classes. (<b>D</b>) Heatmaps showing confidence of predictions made by diverse C vs. NC classifiers for male (M) and female (F) mice treated with CPA, TAA and WY for 3 days (left heatmap) or 14 days (right heatmap). (<b>E</b>) Similar illustration as in (D) showing prediction outcomes of GC vs. NGC classifiers.</p