Aberrant methylation of tRNAs links cellular stress to neuro-developmental disorders.

Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. How post-transcriptional methylation contributes to the human disease is currently unknown. By comparing gene expression data with global cytosine-5 RNA methylomes in patient fibroblasts and NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the angiogenin-mediated endonucleolytic cleavage of transfer RNAs (tRNA) leading to an accumulation of 5' tRNA-derived small RNA fragments. Accumulation of 5' tRNA fragments in the absence of NSun2 reduces protein translation rates and activates stress pathways leading to reduced cell size and increased apoptosis of cortical, hippocampal and striatal neurons. Mechanistically, we demonstrate that angiogenin binds with higher affinity to tRNAs lacking site-specific NSun2-mediated methylation and that the presence of 5' tRNA fragments is sufficient and required to trigger cellular stress responses. Furthermore, the enhanced sensitivity of NSun2-deficient brains to oxidative stress can be rescued through inhibition of angiogenin during embryogenesis. In conclusion, failure in NSun2-mediated tRNA methylation contributes to human diseases via stress-induced RNA cleavage

Liver mitochondria isolated by magnetic beads have higher oxygen consumption rates than mitochondria isolated by differential centrifugation.

<p>Oxygen consumption rates (OCRs) were determined using an XF24 Extracellular Flux Analyzer with the given amount of freshly isolated mitochondria (µg/well), which were purified by <b>A</b> DC or <b>B</b> MB methods. Briefly, succinate was used as a substrate to feed electrons to complex II and basal (state II) respiration was measured. State III respiration was determined in the presence of ADP. Oligomycin (Oligo) addition blocked ATP synthase and so evoked state IVo respiration. Maximum respiration (state uncoupled) was measured in the presence of the uncoupler FCCP. Finally, Antimycin A (Antim) was given to inhibit complex III. Data are represented as means of three independent experiments (5–8 replicates per groups) ± SD.</p

Liver, muscle and brain mitochondria isolated using anti-TOM22 magnetic beads show reliable mitochondrial function.

<p>Oxygen consumption of isolated <b>A</b> liver, <b>B</b> muscle and <b>C</b> brain mitochondria were measured by a Clark electrode. Malate+pyruvate (MP) and malate+glutamate (MG) served as specific substrates for complex I, succinate (S, for complex II and glycerol-3-phosphate (GP) for complex III, respectively. ADP was given to malate+pyruvate to determine state III respiration via complex I. Oligomycin and CCCP were used with succinate to assess state IVo and uncoupled respiration, respectively. The given oxygen consumption units are nmol oxygen/min/mg mitochondria. Data are represented as means of three independent experiments ± SD.</p

Metabolic syndrome and extensive adipose tissue inflammation in morbidly obese Göttingen minipigs

Objective: The worldwide prevalence of obesity has increased to 10% in men and 15% in women and is associated with severe comorbidities such as diabetes, cancer, and cardiovascular disease. Animal models of obesity are central to experimental studies of disease mechanisms and therapeutic strategies. Diet-induced obesity (DIO) models in rodents have provided important insights into the pathophysiology of obesity and, in most instances, are the first in line for exploratory pharmacology studies. To deepen the relevance towards translation to human patients, we established a corresponding DIO model in Göttingen minipigs (GM). Methods: Young adult female ovariectomized GM were fed a high-fat/high-energy diet for a period of 70 weeks. The ration was calculated to meet the requirements and maintain body weight (BW) of lean adult minipigs (L-GM group) or increased stepwise to achieve an obese state (DIO-GM group). Body composition, blood parameters and intravenous glucose tolerance were determined at regular intervals. A pilot chronic treatment trial with a GLP1 receptor agonist was conducted in DIO-GM. At the end of the study, the animals were necropsied and a biobank of selected tissues was established. Results: DIO-GM developed severe subcutaneous and visceral adiposity (body fat >50% of body mass vs. 22% in L-GM), increased plasma cholesterol, triglyceride, and free fatty acid levels, insulin resistance (HOMA-IR >5 vs. 2 in L-GM), impaired glucose tolerance and increased heart rate when resting and active. However, fasting glucose concentrations stayed within normal range throughout the study. Treatment with a long-acting GLP1 receptor agonist revealed substantial reduction of food intake and body weight within four weeks, with increased drug sensitivity relative to observations in other DIO animal models. Extensive adipose tissue inflammation and adipocyte necrosis was observed in visceral, but not subcutaneous, adipose tissue of DIO-GM. Conclusions: The Munich DIO-GM model resembles hallmarks of the human metabolic syndrome with extensive adipose tissue inflammation and adipocyte necrosis reported for the first time. DIO-GM may be used for evaluating novel treatments of obesity and associated comorbidities. They may help to identify triggers and mechanisms of fat tissue inflammation and mechanisms preventing complete metabolic decompensation despite morbid obesity. Keywords: Pig, High fat diet, Obesity, Diabetes, Metabolic syndrome, Adipose tissue inflammatio