Cell autonomous requirement of neurofibromin (Nf1) for postnatal muscle hypertrophic growth and metabolic homeostasis

Background Neurofibromatosis type 1 (NF1) is a multi-organ disease caused by mutations in neurofibromin 1 (NF1). Amongst other features, NF1 patients frequently show reduced muscle mass and strength, impairing patients' mobility and increasing the risk of fall. The role of Nf1 in muscle and the cause for the NF1-associated myopathy are mostly unknown. Methods To dissect the function of Nf1in muscle, we created muscle-specific knockout mouse models for NF1, inactivatingNf1in the prenatal myogenic lineage either under the Lbx1 promoter or under the Myf5 promoter. Mice were analysed during prenatal and postnatal myogenesis and muscle growth. Results Nf1(Lbx1)and Nf1(Myf5)animals showed only mild defects in prenatal myogenesis. Nf1(Lbx1)animals were perinatally lethal, while Nf1(Myf5)animals survived only up to approximately 25 weeks. A comprehensive phenotypic characterization of Nf1(Myf5)animals showed decreased postnatal growth, reduced muscle size, and fast fibre atrophy. Proteome and transcriptome analyses of muscle tissue indicated decreased protein synthesis and increased proteasomal degradation, and decreased glycolytic and increased oxidative activity in muscle tissue. High-resolution respirometry confirmed enhanced oxidative metabolism in Nf1(Myf5)muscles, which was concomitant to a fibre type shift from type 2B to type 2A and type 1. Moreover, Nf1(Myf5)muscles showed hallmarks of decreased activation of mTORC1 and increased expression of atrogenes. Remarkably, loss of Nf1 promoted a robust activation of AMPK with a gene expression profile indicative of increased fatty acid catabolism. Additionally, we observed a strong induction of genes encoding catabolic cytokines in muscle Nf1(Myf5)animals, in line with a drastic reduction of white, but not brown adipose tissue. Conclusions Our results demonstrate a cell autonomous role for Nf1 in myogenic cells during postnatal muscle growth required for metabolic and proteostatic homeostasis. Furthermore, Nf1 deficiency in muscle drives cross-tissue communication and mobilization of lipid reserves

Low steady-state oxidative stress inhibits adipogenesis by altering mitochondrial dynamics and decreasing cellular respiration

Adipogenesis is a fundamental process of white adipose tissue function, supporting lipid storage and release, while avoiding its spillover and ectopic accumulation in tissues and organs. During aging adipogenesis is impaired and among other factors, oxidative stress contributes to this process. Adipogenesis requires functional and dynamic mitochondria; however, this organelle itself becomes dysfunctional during aging and accounts for most of reactive oxygen species (ROS) production. Here, we evaluated whether oxidative stress impairs adipogenesis through functional impairment of mitodynamics by utilizing hyperoxia as a continuous source of oxidative stress while maintaining cellular viability. This negatively impacted mitochondrial function, including respiration and dynamics and ultimately blocked adipogenesis. Interestingly, this state was reversible by using the antidiabetic drug, Rosiglitazone, which reduced oxidative stress, restored mitochondrial dynamics and respiration and augmented adipogenesis. Moreover, in vitro results were in agreement with in vivo models of oxidative stress and aging, in which mice depleted of the superoxide dismutase enzyme 1 (SOD1) and old wild-type C57BL/6JRj mice demonstrated the same trend of adipogenic potential. Importantly, in humans the results follow the same pattern, showing a downregulation of adipogenic markers during aging. Since the levels of oxidative stress and peripheral insulin resistance increase with age, while adipogenesis decreases during aging, our model helps to understand a possible way to overcome physiologically low, steady stress conditions and restore adipogenesis, avoiding accumulation of deleterious hypertrophic adipocytes in favor of beneficial hyperplasia

Reversible gene knockdown in mice using a tight, inducible shRNA expression system

RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics. Techniques allowing to control time and degree of gene silencing in vivo, however, are still lacking. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. To demonstrate the feasibility of our tool, a mouse model of reversible insulin resistance was generated by expression of an insulin receptor (Insr)-specific shRNA. Upon induction, mice develop severe hyperglycemia within seven days. The onset and progression of the disease correlates with the concentration of doxycycline, and the phenotype returns to baseline shortly after withdrawal of the inductor. On a broad basis, this approach will enable new insights into gene function and molecular disease mechanisms

Insulin and insulin-like growth factor 1 receptors are required for normal expression of imprinted genes

In addition to signaling through the classical tyrosine kinase pathway, recent studies indicate that insulin receptors (IRs) and insulin-like growth factor 1 (IGF1) receptors (IGF1Rs) can emit signals in the unoccupied state through some yet-to-be-defined noncanonical pathways. Here we show that cells lacking both IRs and IGF1Rs exhibit a major decrease in expression of multiple imprinted genes and microRNAs, which is partially mimicked by inactivation of IR alone in mouse embryonic fibroblasts or in vivo in brown fat in mice. This down-regulation is accompanied by changes in DNA methylation of differentially methylated regions related to these loci. Different from a loss of imprinting pattern, loss of IR and IGF1R causes down-regulated expression of both maternally and paternally expressed imprinted genes and microRNAs, including neighboring reciprocally imprinted genes. Thus, the unoccupied IR and IGF1R generate previously unidentified signals that control expression of imprinted genes and miRNAs through transcriptional mechanisms that are distinct from classical imprinting control

Schematic representation of the inducible system and gene targeting strategy

<p><b>Copyright information:</b></p><p>Taken from "Reversible gene knockdown in mice using a tight, inducible shRNA expression system"</p><p></p><p>Nucleic Acids Research 2007;35(7):e54-e54.</p><p>Published online 21 Mar 2007</p><p>PMCID:PMC1874634.</p><p>© 2007 The Author(s)</p> Principle of tetR-mediated control of shRNA expression. Transcription of the RNA polymerase III-dependent promoter is blocked in cells expressing the tet repressor (tetR). Upon induction by doxycycline, tetR is removed from the tet-operator sequences (tetO) inserted into the promoter, allowing transcription of shFluc. ShRNA expression leads to RNAi-mediated knockdown of the target gene firefly luciferase. Renilla luciferase is used for reference to quantify firefly luciferase activity. Scheme of the targeting strategy. ShRNA and reporter constructs were independently inserted into the locus by homologous recombination in ES cells. Genes encoding the (Rluc) and firefly luciferases (Fluc) along with an adenovirus splice acceptor sequence and a polyadenylation signal (pA) were placed downstream of the endogenous promoter. The Fluc-specific shRNA is expressed under the control of the U6-tet or H1-tet promoter, and terminated by five thymidines (shRNA). The -sites flanking the shRNA expression cassettes were used to generate a negative control through cre-mediated recombination in ES cells. Southern blot analysis of genomic DNA from transfected ES cell clones containing the shRNA- (lane #1 and #2) or the reporter-constructs (lanes #3 and #4). Homologous recombination at the locus is detectable by using EcoRV-digested genomic DNA and probe 1, resulting in a 11.7 kb band for the wt and a 2.5 kb band for targeted allele. E: EcoRV; X: XbaI; neo: FRT-flanked neomycin resistance gene; hyg: FRT-flanked hygromycin resistance gene. () Western blot analysis from protein extracts of ES cells expressing either the wt tetR or the itetR using tetR- or β-Actin-specific antiserum. Control: protein extracts from wt ES cells

Efficiency of H1/U6-shRNA-mediated firefly luciferase (Fluc) knockdown in mice expressing the codon-optimized tetR

<p><b>Copyright information:</b></p><p>Taken from "Reversible gene knockdown in mice using a tight, inducible shRNA expression system"</p><p></p><p>Nucleic Acids Research 2007;35(7):e54-e54.</p><p>Published online 21 Mar 2007</p><p>PMCID:PMC1874634.</p><p>© 2007 The Author(s)</p> Each configuration (control, H1- shRNA, U6 shRNA) was analyzed using two to four mice at the age of 8–10 weeks, respectively. Percentages of shRNA-mediated repression of firefly luciferase activity with standard error of the mean are shown for untreated controls (gray bars) and in animals upon 10 days feeding with 2 mg/ml doxycycline in the drinking water (white bars). In negative control animals (black bars), the shRNA expression cassettes are removed through cre-mediated recombination. Relative values of relative firefly luciferase expression level in different organs were calculated by using luciferases activities for reference. () H1 promoter-driven shRNA expression. () shRNA expression through the U6 promoter