Identifizierung, Genkartierung, und Charakterisierung von ENU-induzierten mutanten Mauslinien für Knochenkrankheiten des Menschen

Abstract

INTRACELLULAR ACCUMULATION OF ABNORMAL PRO-α1(I) CAUSES ER STRESS AND OSTEOBLAST APOPTOSIS IN A NEW MOUSE MODEL FOR TYPE II/III/IV OSTEOGENESIS IMPERFECTA Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here we describe a new mouse model for osteogenesis imperfecta termed Aga2 (abnormal gait 2) which was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability including reduced bone mass, multiple fractures and early lethality. The causal gene was mapped to chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1) gene as the cause of the disorder. Heterozygous animals had markedly increased bone turnover and a disrupted native collagen network associated with microfractures in vivo. Further studies showed that abnormal pro-α1(I) chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an ER stress-specific unfolded protein response (UPR) involving upregulation of BiP, Hsp47 and Gadd153 with caspases 12 and 3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for osteogenesis imperfecta, and identified a role for intracellular modulation of the ER stress-associated UPR machinery towards osteoblast apoptosis during the pathogenesis of disease.

CHARACTERIZATION AND MAPPING OF ALI34: A ENU-DERIVED MURINE MODEL DEPICTING FEATURES OF OSTEOARTHRITIS AND OSTEOCHONDRODYSPLASIA The Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program was recently utilized to identify and characterize new genes and alleles that regulate skeletal development and homeostasis in mouse. Subsequently, the Ali34 autosomal dominant mutant line was isolated depicting several features of osteoarthritis (OA) and osteochondrodysplasia. OA is the most common type of arthritis affecting up to 30 % of people over age 55. Ali34/+ and Ali34/Ali34 skeletal phenotypes were characterized, and the genetic mapping of the Ali34 locus was reported. Skeletal morphology was assessed via μCT and alizarin red/alcian blue staining. Dynamic histomorphometry was performed via tetracycline/calcin double-labeling. Proteoglycans were assessed using safranin O. Chondromodulin I (ChM-I), an inhibitor of angiogenesis marker, was detected via immunohistochemistry. Bone and body composition parameters were measured via pDXA and pQCT. Linkage analysis was conducted via the outcross-backcross (F1 X P) breeding strategy utilizing a high throughput SNP haplotyping platform. 12-week-old Ali34/+ animals exhibit shortened tibiae and femora, and anklyosis and osteophytes affecting the knee joint region. Both periarticular and epiphysial bones displayed subchondral sclerosis, and mineral components were evident within the articular space. A decrease in the rate of new trabecular bone formation was accompanied by a ~13 % reduction in vBMD (295.4 ± 15.4 mg/cm3 control [n = 10] and 258.3 ± 30.5 mg/cm3 Ali34/+ [n = 8]; p = 0.001) at the femoral metaphysis. Abnormalities of the growth plate were apparent as absence of the resting zone of chondrocytes, and disorganization of the avascular zone as shown by ChM-I reactivity. In Ali34 articular cartilage, the transitional zone was absent, and erosion of matrix was evident without fibrillation. The articular capsule contained increased mesenchyme cells, and tendon metaplasia was obvious. pDXA results depicted increased lean mass (~21 %, p = 0.001, n = 6) and lowered fat mass (~37.5 %, p = 0.001, n = 6) in Ali34 males. Ali34/Ali34 animals died embryonically between E16 - 19, and displayed both caudal calvarial agenesis and deviated vertebral disks. Chromosomal mapping studies revealed Ali34 linkage on mouse chromosome 6, and subsequently fine mapping and candidate gene sequencing investigations identified a mutation in the Plxnd1 (plexin D1) gene crucial for vasculogenesis and angiogenesis maintenance. Our observations demonstrated that the Ali3 4 locus played a key role in regulating the development of the epiphysial growth plate and knee joints, and proper development of skeletal elements derived from the (paraxial) mesoderm and neural crest . Lastly, based on the Ali34 phenotype and causal gene product, the hereditary congenital facial paresis (HCFP; OMIM 601471) susceptibility locus may reflect anomalies in the plexin family of genes. The Ali34 line represents a valuable animal model with which to investigate the (early) pathogenesis of OA and osteochondrodysplasia.

Intrazellulaere Akkumulation von abnormalem Pro-Collagen a1 verursacht ER - Stress und Osteoblast Apoptosis in einem neuen Mausmodel fuer Typ II Osteogenesis Imperfecta OI ist eine Erbkrankheit, die durch Knochenschwaeche, Knochenbrueche und Osteoporose gekennzeichnet ist. Verursacht wird diese Krankheit durch einen Gendefekt im Typ I Collagen Gen. In dieser Studie wurde nach einem ENU-Mutagenese Screen ein neues Mausmodel (Aga2) fuer OI identifiziert und untersucht. Mutante Mauese der Aga2 Linie weisen phenotypische Variabilitaet, eine reduzierte Knochenmasse, Knochenbrueche sowie embryonale Lethalitaet auf. Typ I Collagen wurde kartiert und konnte Chromosom 11 zugeordnet werden. Das Gen zeigt einen frameshift im C-terminalen Bereich von Aga2 Maeusen. Heterozygote Tiere zeigen einen signifikant erhoehten Knochenabbau und ein abnormales Kollagennetzwerk in vivo. Mutierte Kollagenpeptidketten akkumulieren intrazellulaer und verursachen eine Fehlerhafte Sezernierung von dermalen Fibroblasten in die Extrazellulaermatrix. Zudem konnte dies mit ER-Stress spezifischer Proteinedenaturierung korreliert werden, welche ueber BiP, Hsp47, Gadd153 und den Caspasen 3 und 12 vermittelt wird und zur Apoptose von Osteoblasten in vivo und in vitro fuehrt.

Charakterisierung und Kartierung von Ali34: Ein Mausmodel aus einem ENU-Mutagense Screen fuer Osteoarthritis und Osteochondrodysplasia Im kuerzlich erfolgreich durchgefuehrten ENU Mutagenese Screen am Helmholtz-Zentrum Muenchen konnten mehrere neue Mausmodelle fuer die Untersuchung von Skelettentwicklung und Homeostase identifiziert werden. Eine identifizierte Mutante (Ali34) ist autosomal dominant und weist mehrere Merkmale auf, die in auch in Patienten mit Osteoarthritis (OA) und Osteochondrodysplasia zu finden sind. Untersuchungen der Knochenbeschaffenheit sowie der Knochendichte zeigten Abnormalitaeten in der Knochenentwicklung und Knochenhomeostase. Genkartierung zeigte die Lokalisation von Ali34 auf Chr 6, nahe des Plexin d1 lokus, welches fuer Vaskularisierung und Angiogenese bedeutend ist. In dieser Studie konnte gezeigt werden, dass Ali34 eine wichtige Rolle bei der Entwicklung der Epiphysenwachstumplatte, der Kniegelenke sowie der Entwicklung von skeletalen Elementen, die aus Mesoderm und Neuralleistenzellen stammen spielt.