A mouse model for embryonal tumors with multilayered rosettes uncovers the therapeutic potential of Sonic-hedgehog inhibitors.

Embryonal tumors with multilayered rosettes (ETMRs) have recently been described as a new entity of rare pediatric brain tumors with a fatal outcome. We show here that ETMRs are characterized by a parallel activation of Shh and Wnt signaling. Co-activation of these pathways in mouse neural precursors is sufficient to induce ETMR-like tumors in vivo that resemble their human counterparts on the basis of histology and global gene-expression analyses, and that point to apical radial glia cells as the possible tumor cell of origin. Overexpression of LIN28A, which is a hallmark of human ETMRs, augments Sonic-hedgehog (Shh) and Wnt signaling in these precursor cells through the downregulation of let7-miRNA, and LIN28A/let7a interaction with the Shh pathway was detected at the level of Gli mRNA. Finally, human ETMR cells that were transplanted into immunocompromised host mice were responsive to the SHH inhibitor arsenic trioxide (ATO). Our work provides a novel mouse model in which to study this tumor type, demonstrates the driving role of Wnt and Shh activation in the growth of ETMRs and proposes downstream inhibition of Shh signaling as a therapeutic option for patients with ETMRs.

Dissociation of somatic growth, time of sexual maturity, and life expectancy by overexpression of an RGD-deficient IGFBP-2 variant in female transgenic mice.

Impaired growth is often associated with an extension of lifespan. However, the negative correlation between somatic growth and life expectancy is only true within, but not between, species. This can be observed because smaller species have, as a rule, a shorter lifespan than larger species. In insects and worms, reduced reproductive development and increased fat storage are associated with prolonged lifespan. However, in mammals the relationship between the dynamics of reproductive development, fat metabolism, growth rate, and lifespan are less clear. To address this point, female transgenic mice that were overexpressing similar levels of either intact (D-mice) or mutant insulin-like growth factor-binding protein-2 (IGFBP-2) lacking the Arg-Gly-Asp (RGD) motif (E- mice) were investigated. Both lines of transgenic mice exhibited a similar degree of growth impairment (-9% and -10%) in comparison with wild-type controls (C-mice). While in D-mice, sexual maturation was found to be delayed and life expectancy was significantly increased in comparison with C-mice, these parameters were unaltered in E-mice in spite of their reduced growth rate. These observations indicate that the RGD-domain has a major influence on the pleiotropic effects of IGFBP-2 and suggest that somatic growth and time of sexual maturity or somatic growth and life expectancy are less closely related than thought previously.

Efavirenz Causes Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy in Endothelial Cells.

The non-nucleoside reverse transcriptase inhibitor efavirenz is a widely prescribed antiretroviral drug used in combined antiretroviral therapy. Despite being an essential and life-saving medication, the required lifelong use of HIV drugs has been associated with a variety of adverse effects, including disturbances in lipid metabolism and increased cardiovascular risk. Efavirenz belongs to those HIV drugs for which cardiovascular and endothelial dysfunctions have been reported. It is here shown that elevated concentrations of efavirenz can inhibit endothelial meshwork formation on extracellular matrix gels by normal and immortalized human umbilical vein cells. This inhibition was associated with an increase in oxidative stress markers, endoplasmic reticulum (ER) stress markers, and autophagy. Induction of ER stress occurred at pharmacologically relevant concentrations of efavirenz and resulted in reduced proliferation and cell viability of endothelial cells, which worsened in the presence of elevated efavirenz concentrations. In combination with the HIV protease inhibitor nelfinavir, both oxidative stress and ER stress became elevated in endothelial cells. These data indicate that pharmacologically relevant concentrations of efavirenz can impair cell viability of endothelial cells and that these effects may be aggravated by either elevated concentrations of efavirenz or by a combined use of efavirenz with other oxidative stress-inducing medications.

Protein O-mannosylation is crucial for E-cadherin-mediated cell adhesion.

In recent years protein O-mannosylation has become a focus of attention as a pathomechanism underlying severe congenital muscular dystrophies associated with neuronal migration defects. A key feature of these disorders is the lack of O-mannosyl glycans on α-dystroglycan, resulting in abnormal basement membrane formation. Additional functions of O-mannosylation are still largely unknown. Here, we identify the essential cell-cell adhesion glycoprotein epithelial (E)-cadherin as an O-mannosylated protein and establish a functional link between O-mannosyl glycans and cadherin-mediated cell-cell adhesion. By genetically and pharmacologically blocking protein O-mannosyltransferases, we found that this posttranslational modification is essential for preimplantation development of the mouse embryo. O-mannosylation-deficient embryos failed to proceed from the morula to the blastocyst stage because of defects in the molecular architecture of cell-cell contact sites, including the adherens and tight junctions. Using mass spectrometry, we demonstrate that O-mannosyl glycans are present on E-cadherin, the major cell-adhesion molecule of blastomeres, and present evidence that this modification is generally conserved in cadherins. Further, the use of newly raised antibodies specific for an O-mannosyl-conjugated epitope revealed that these glycans are present on early mouse embryos. Finally, our cell-aggregation assays demonstrated that O-mannosyl glycans are crucial for cadherin-based cell adhesion. Our results redefine the significance of O-mannosylation in humans and other mammals, showing the immense impact of cadherins on normal as well as pathogenic cell behavior.

Lack of Pur-alpha alters postnatal brain development and causes megalencephaly.

Pur-alpha (Purα) plays an important role in a variety of cellular processes including transcriptional regulation, cell proliferation and oncogenic transformation. To better understand the role of Purα in the developing and mature brain, we generated Purα-deficient mice, which we were able to raise to the age of six months. Purα(-/-) mice were born with no obvious pathological condition. We obtained convincing evidence that lack of Purα prolongs the postnatal proliferation of neuronal precursor cells both in the hippocampus and in the cerebellum, however, without affecting the overall number of postmitotic neurons. Independent of these findings, we observed alterations in the expression and distribution of the dendritic protein MAP2, the translation of which has been proposed previously to be Purα-dependent. At the age of 2 weeks, Purα(-/-) mice generated a continuous tremor which persisted throughout lifetime. Finally, adult Purα(-/-) mice displayed a megalencephaly and histopathological findings including axonal swellings and hyperphosphorylation of neurofilaments. Our studies underline the importance of Purα in the proliferation of neuronal precursor cells during postnatal brain development and suggest a role for Purα in the regulation of the expression and cellular distribution of dendritic and axonal proteins. Since recent studies implicate a link between Purα and the fragile X tremor/ataxia syndrome, our Purα(-/-) mouse model will provide new opportunities for understanding the mechanisms of neurodegeneration.

Insulin glargine and NPH insulin increase to a similar degree epithelial cell proliferation and aberrant crypt foci formation in colons of diabetic mice.

The insulin analog glargine has a higher binding affinity than regular insulin for the insulin-like growth factor 1 receptor in vitro, raising questions about increased mitogenicity in vivo. Observational studies in humans have recently reported a potential differential association between insulin glargine and malignancies, but available evidence remains inconclusive. We directly compared glargine vs. neutral protamine Hagedorn (NPH) insulin's effects on cell proliferation in colonic mucosa and on formation of aberrant crypt foci in diabetic mice, i.e., early stages of colorectal cancer development. Mice (BKS.Cg-+Lepr(db)/+Lepr(db)/OlaHsd) were treated with insulin glargine (G), NPH insulin (NPH), or saline (NaCl). We assessed epithelial proliferation after long-term insulin treatment (18 weeks) by 5-bromo-2'-deoxyuridine and Ki67 staining and analyzed the formation of aberrant crypt foci (ACF) in mice treated with insulin glargine or NPH insulin or 10 weeks after initiation with 1,2-dimethylhydrazine. Insulin glargine treatment did not result in significantly different epithelial colonic proliferation compared to NPH insulin (G, 137 ± 22; NPH, 136 ± 15; NaCl, 100 ± 20 (relative proliferation index)), but both insulin-treated groups of mice had a higher proliferation index compared to the NaCl control group (p<0.001). Similarly, we observed no difference in ACF formation between glargine- and NPH-insulin-treated mice (G, 132 ± 12; NPH, 138 ± 9; NaCl, 100 ± 7 (relative number of ACF)), but ACF formation was significantly higher in insulin-treated mice than in NaCl-treated control mice (p=0.001). Chronic insulin treatment results in higher colonic epithelial proliferation and ACF formation, but the use of insulin glargine vs. NPH insulin is not associated with increased risk.

Specific transgene expression in mouse pancreatic beta-cells under the control of the porcine insulin promoter.

The availability of regulatory sequences directing tissue-specific expression of transgenes in genetically modified mice and large animals is a prerequisite for the development of adequate models for human diseases. The rat insulin 2 gene (Ins2) promoter, widely used to achieve transgene expression in pancreatic beta-cells of mice, also directs expression to extrapancreatic tissues and performs poorly in isolated pancreatic islets of human, mouse, and pig. To evaluate whether the full 5' untranslated region (UTR) of the porcine insulin gene (INS) confers robust and specific expression in beta-cells we generated an expression cassette containing 1500bp of the porcine INS 5' UTR and the 3' UTR of the bovine growth hormone gene (GH). The cassette was designed to allow easy exchange of the sequences to be expressed and easy removal of the vector backbone from the expression cassette. To evaluate the properties of the cassette, we initially inserted a cDNA encoding human betacellulin, a growth factor known to affect structural and functional parameters of beta-cells. After confirming the functionality and specificity of the construct in vitro, transgenic mouse lines were generated by pronuclear DNA microinjection. Using RT-PCR, immunohistochemistry and immunofluorescence, we show that transgenic mice expressed human betacellulin exclusively in beta-cells. Confirming the proposed insulinotropic effect of betacellulin, transgenic mice showed improved glucose tolerance. We conclude that the newly designed expression cassette containing 1500bp of the porcine insulin promoter 5' UTR confers robust and specific transgene expression to beta-cells in vitro and in transgenic mice.

Scrapie-infected transgenic mice expressing a laminin receptor decoy mutant reveal a prolonged incubation time associated with low levels of PrPres.

The 37-kDa/67-kDa laminin receptor (LRP/LR) was identified as a cell surface receptor for prion proteins. The laminin receptor mutant LRP102-295::FLAG interfered with PrP(Sc) propagation in murine neuronal cells presumably acting as a decoy in a transdominant negative fashion by trapping PrP molecules in the extracellular matrix. Here, we generated hemizygous transgenic mice expressing LRP102-295::FLAG in the brain. Scrapie-infected transgenic mice exhibit a significantly prolonged incubation time in comparison to scrapie-infected wild-type (FVB) mice. At the terminal stage, transgenic mice revealed significantly reduced proteinase-K-resistant PrP levels by 71% compared to wild-type mice. Our results recommend the laminin receptor decoy mutant as an alternative therapeutic tool for treatment of transmissible spongiform encephalopathies.

IGFBP-2 overexpression reduces the appearance of dysplastic aberrant crypt foci and inhibits growth of adenomas in chemically induced colorectal carcinogenesis.

Colon cancer patients frequently show increased levels of serum insulin-like growth factor-binding protein-2 (IGFBP-2), however, the pathogenetic relevance of this phenomenon for colorectal cancer is unclear. Therefore, we have used IGFBP-2 transgenic animals which overexpress IGFBP-2 systemically and locally in the intestine to study its role in chemically induced colorectal carcinogenesis. Mice received intraperitoneal injections of 1,2-dimethylhydrazine (DMH) (40 mg/kg body weight) once a week for 6 weeks to selectively induce aberrant crypt foci (ACF) and tumors in the colon. While tumor incidence was comparable in transgenic and control mice, the volume of adenomas in IGFBP-2 transgenic mice was reduced more than 2-fold. Furthermore, serum IGFBP-2 levels negatively correlated with tumor volume in the IGFBP-2 transgenic group. Histological examination showed that IGFBP-2 transgenic mice developed significantly less dysplastic ACF with a high potential to progress to advanced stages. The reduced tumor volume in IGFBP-2 transgenic animals was due to significantly reduced proliferative capacity, evidenced by a lower proportion of cells positive for Ki67. Our results demonstrate for the first time in an experimental model that IGFBP-2 overabundance prior to the onset and during colorectal carcinogenesis reduces tumor growth by inhibition of cell proliferation.

Systemic overexpression of growth hormone (GH) in transgenic FVB/N inbred mice: an optimized model for holistic studies of molecular mechanisms underlying GH-induced kidney pathology.

Transgenic mice overexpressing growth hormone (GH) display a plethora of phenotypic alterations and provide unique models for studying and influencing consequences of chronic GH excess. Since the first report on GH transgenic mice was published in 1982, many different mouse models overexpressing GH from various species at different levels and with different tissue specificities were established, most of them on random-bred or hybrid genetic background. We have generated a new transgenic mouse model on FVB/N inbred background, expressing bovine (b) GH under the control of the chicken beta-actin promoter (cbetaa). cbetaa-bGH transgenic mice exhibit ubiquitous expression of bGH mRNA and protein and circulating bGH levels in the range of several microg/ml, resulting in markedly stimulated growth and the characteristic spectrum of pathological lesions which were described in previous GH overexpressing mouse models. Importantly, a consistent sequence of renal alterations is observed, mimicking progressive kidney disease in human patients. The novel, genetically standardized GH transgenic mouse model is ideal for holistic transcriptome and proteome studies aiming at the identification of the molecular mechanisms underlying GH-induced pathological alterations especially in the kidney. Moreover, genetically defined cbetaa-bGH mice facilitate random mutagenesis screens for modifier genes which influence the effects of chronic GH excess and associated pathological lesions.

Hepatic selenoprotein P (SePP) expression restores selenium transport and prevents infertility and motor-incoordination in Sepp-knockout mice.

SePP (selenoprotein P) is central for selenium transport and distribution. Targeted inactivation of the Sepp gene in mice leads to reduced selenium content in plasma, kidney, testis and brain. Accordingly, activities of selenoenzymes are reduced in Sepp(-/-) organs. Male Sepp(-/-) mice are infertile. Unlike selenium deficiency, Sepp deficiency leads to neurological impairment with ataxia and seizures. Hepatocyte-specific inactivation of selenoprotein biosynthesis reduces plasma and kidney selenium levels similarly to Sepp(-/-) mice, but does not result in neurological impairment, suggesting a physiological role of locally expressed SePP in the brain. In an attempt to define the role of liver-derived circulating SePP in contrast with locally expressed SePP, we generated Sepp(-/-) mice with transgenic expression of human SePP under control of a hepatocyte-specific transthyretin promoter. Secreted human SePP was immunologically detectable in serum from SEPP1-transgenic mice. Selenium content and selenoenzyme activities in serum, kidney, testis and brain of Sepp(-/-;SEPP1) (SEPP1-transgenic Sepp(-/-)) mice were increased compared with Sepp(-/-) controls. When a selenium-adequate diet (0.16-0.2 mg/kg of body weight) was fed to the mice, liver-specific expression of SEPP1 rescued the neurological defects of Sepp(-/-) mice and rendered Sepp(-/-) males fertile. When fed on a low-selenium diet (0.06 mg/kg of body weight), Sepp(-/-;SEPP1) mice survived 4 weeks longer than Sepp(-/-) mice, but ultimately developed the neurodegenerative phenotype. These results indicate that plasma SePP derived from hepatocytes is the main transport form of selenium supporting the kidney, testis and brain. Nevertheless, local Sepp expression is required to maintain selenium content in selenium-privileged tissues such as brain and testis during dietary selenium restriction.

Effect of macrophage ApoE on atherosclerosis in LDL-receptor deficient mice.

Apolipoprotein E (ApoE) plays an important role in the development of atherosclerosis. Previous studies provide evidence for an atheroprotective role of ApoE in mouse models on the ApoE deficient (ApoE-/-) background. However, it is not clear whether this is also true on the LDL-receptor deficient (LDLR-/-) background. Transgenic mice carrying hApoE coding sequences in a chicken lysozyme expression cassette were generated. Transgene expression was directed into macrophages, expressing low levels of hApoE. Expression of the hApoE transgene was not sufficient to correct hypercholesterolemia. However, lesion area at the brachiocephalic artery (BCA) was significantly reduced (-72%) in female hApoE transgenic mice on the LDLR-/- background. This was associated with increased cholesterol efflux in macrophages of transgenic animals on the ApoE-/- background. We conclude that over-expression of ApoE in macrophages might be useful as a therapeutic principle for the prevention of atherosclerosis.

Adult scl+/+ murine hemangioblasts persist in allogeneic mutant blastocysts but fail to rescue the scl-/- phenotype.

Isolated and expanded scl (+) adult murine progenitors show a strong endothelial and hematopoietic differentiation potential and have been considered to be the adult equivalent of the hemangioblast. These unique cells may provide effective therapeutic approaches to tissue damage resulting from hypoxemia or chronic ischemia. Here, we study the fate of adult scl (+/+) during development and their ability to reverse genetic defects in scl expression. scl (+/+) adult stem cells (clone RM26) did not persist during embryonic development after injection into blastocysts of allogeneic wild-type mice on day E 3.5. However, GFP(+)-marked scl (+/+) cells were detected in all possible genotypes from allogeneic scl (+/+) intercrosses (scl (+/+), scl (+/-), scl (-/-) on day E 9.5 after the cloned cells were injected into scl-mutant blastocysts on day E 3.5. Nevertheless, there was no indication of phenotypic rescue of the mutant blastocysts despite the continued presence of scl (+/+) RM26 cells in the allogeneic embryonic environment. The results show that differentiated stem cells providing a defective gene may exert effects during development when there is a reparative demand, but they are not capable of reversing the effects of a mutant phenotype during embryonic development. These effects should be considered when evaluating the efficacy of stem cells for therapeutic reversal of inborn errors of development.

A 470 bp WAP-promoter fragment confers lactation independent, progesterone regulated mammary-specific gene expression in transgenic mice.

The ability of a 470 bp sub-fragment of the murine whey acidic protein (WAP) promoter in the context of a retroviral expression plasmid to direct gene expression to mammary epithelial cells was analysed in a number of independent transgenic mouse lines. In contrast to previous findings with the genuine 2.5 kb promoter fragment, our studies revealed a highly mammary gland-specific expression detectable only in non-lactating animals. This suggested a mainly progesterone-regulated activity of the short fragment. Therefore, transgene expression was examined in the progesterone-determined estrous cycle and during pregnancy. In accordance with in vitro data from stably transfected cell lines, in both situations expression was upregulated at stages associated with high progesterone levels. Taken together these data provide deeper insight into WAP-promoter regulation and stress the usefulness of the shortened fragment for a lactation independent mammary-targeted expression.

Targeted disruption of the Walker-Warburg syndrome gene Pomt1 in mouse results in embryonic lethality.

O-mannosylation is an important protein modification in eukaryotes that is initiated by an evolutionarily conserved family of protein O-mannosyltransferases. The first mammalian protein O-mannosyltransferase gene described was the human POMT1. Mutations in the hPOMT1 gene are responsible for Walker-Warburg syndrome (WWS), a severe recessive congenital muscular dystrophy associated with defects in neuronal migration that produce complex brain and eye abnormalities. During embryogenesis, the murine Pomt1 gene is prominently expressed in the neural tube, the developing eye, and the mesenchyme. These sites of expression correlate with those in which the main tissue alterations are observed in WWS patients. We have inactivated a Pomt1 allele by gene targeting in embryonic stem cells and produced chimeras transmitting the defect allele to offspring. Although heterozygous mice were viable and fertile, the total absence of Pomt1(-/-) pups in the progeny of heterozygous intercrosses indicated that this genotype is embryonic lethal. An analysis of the mutant phenotype revealed that homozygous Pomt1(-/-) mice suffer developmental arrest around embryonic day (E) 7.5 and die between E7.5 and E9.5. The Pomt1(-/-) embryos present defects in the formation of Reichert's membrane, the first basement membrane to form in the embryo. The failure of this membrane to form appears to be the result of abnormal glycosylation and maturation of dystroglycan that may impair recruitment of laminin, a structural component required for the formation of Reichert's membrane in rodents. The targeted disruption of mPomt1 represents an example of an engineered deletion of a known glycosyltransferase involved in O-mannosyl glycan synthesis.

Knock-down of the 37-kDa/67-kDa laminin receptor in mouse brain by transgenic expression of specific antisense LRP RNA.

The 37-kDa/67-kDa laminin receptor (LRP/LR) plays a major role in the propagation of PrPSc, the abnormal form of the prion protein. In order to ablate the expression of LRP/LR in mouse brain we generated transgenic mice ectopically expressing antisense LRP RNA in the brain under control of the neuron-specific enolase (NSE) promoter. Hemizygous transgenic mice TgN(NSEasLRP)2 showed a significant reduction of LRP/LR protein levels in hippocampal and cerebellar brain regions. These mice might act as powerful tools to investigate the role of the laminin receptor in scrapie pathogenesis.

Polyclonal anti-PrP auto-antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation in prion-infected cells.

Prion diseases are neurodegenerative infectious disorders for which no prophylactic regimens are known. In order to induce antibodies/auto-antibodies directed against surface-located PrP(c), we used a covalently linked dimer of mouse prion protein expressed recombinantly in Escherichia coli. Employing dimeric PrP as an immunogen we were able to effectively overcome autotolerance against murine PrP in PrP wild-type mice without inducing obvious side effects. Treatment of prion-infected mouse cells with polyclonal anti-PrP antibodies generated in rabbit or auto-antibodies produced in mice significantly inhibited endogenous PrP(Sc) synthesis. We show that polyclonal antibodies are binding to surface-located PrP(c), thereby interfering with prion biogenesis. This effect is much more pronounced in the presence of full IgG molecules, which, unlike Fab fragments, seem to induce a significant cross-linking of surface PrP. In addition, we found immune responses against different epitopes when comparing antibodies induced in rabbits and PrP wild-type mice. Only in the auto-antibody situation in mice an immune reaction against a region of PrP is found that was reported to be involved in the PrP(Sc) conversion process. Our data point to the possibility of developing means for an active immunoprophylaxis against prion diseases.

Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect.

Megalin is an endocytic receptor highly expressed in the proximal tubules of the kidney. Recently, we demonstrated that this receptor is essential for the renal uptake and conversion of 25-OH vitamin D3 to 1,25-(OH)2 vitamin D3, a central step in vitamin D and bone metabolism. Unfortunately, the perinatal lethality of the conventional megalin knockout mouse model precluded the detailed analysis of the significance of megalin for calcium homeostasis and bone turnover in vivo. Here, we have generated a new mouse model with conditional inactivation of the megalin gene in the kidney by using Cre recombinase. Animals with a renal-specific receptor gene defect were viable and fertile. However, lack of receptor expression in the kidney results in plasma vitamin D deficiency, in hypocalcemia and in severe bone disease, characterized by a decrease in bone mineral content, an increase in osteoid surfaces, and a lack of mineralizing activity. These features are consistent with osteomalacia (softening of the bones) as a consequence of hypovitaminosis D and demonstrate the crucial importance of the megalin pathway for systemic calcium homeostasis and bone metabolism.