Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease

The scurfy mutant mouse strain suffers from a fatal lymphoproliferative disease leading to early death within 3–4 wk of age. A frame-shift mutation of the forkhead box transcription factor Foxp3 has been identified as the molecular cause of this multiorgan autoimmune disease. Foxp3 is a central control element in the development and function of regulatory T cells (T reg cells), which are necessary for the maintenance of self-tolerance. However, it is unclear whether dysfunction or a lack of T reg cells is etiologically involved in scurfy pathogenesis and its human correlate, the IPEX syndrome. We describe the generation of bacterial artificial chromosome–transgenic mice termed “depletion of regulatory T cell” (DEREG) mice expressing a diphtheria toxin (DT) receptor–enhanced green fluorescent protein fusion protein under the control of the foxp3 gene locus, allowing selective and efficient depletion of Foxp3+ T reg cells by DT injection. Ablation of Foxp3+ T reg cells in newborn DEREG mice led to the development of scurfy-like symptoms with splenomegaly, lymphadenopathy, insulitis, and severe skin inflammation. Thus, these data provide experimental evidence that the absence of Foxp3+ T reg cells is indeed sufficient to induce a scurfy-like phenotype. Furthermore, DEREG mice will allow a more precise definition of the function of Foxp3+ T reg cells in immune reactions in vivo

No recombination of mtDNA after heteroplasmy for 50 generations in the mouse maternal germline

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Grain legumes in mixed stands with false flax (Camelina sativa) in organic farming under Pannonian site conditions

In drei aufeinander folgenden Versuchsjahren wurden die Körnerleguminosen Linse (Lens culinaris), Platterbse (Lathyrus sativus) und Futtererbse (Pisum sativum) als Reinsaat und in unterschiedlichen Mischungsverhältnissen gemeinsam mit Leindotter (Camelina sativa) in einem Feldversuch auf den ökologisch bewirtschafteten Versuchsflächen der Universität für Bodenkultur Wien im pannonischen Klimagebiet geprüft. Ziel dieses Versuchs war zu überprüfen, ob unter trockenen Standortbedingungen eine Körnerleguminosen-Leindotter-Mischkultur trotz der erhöhten Konkurrenzsituation zwischen den Mischungspartnern vorteilhaft ist. Dafür wurden die optimalen Saatmengen von Leindotter und Körnerlegumi­nosen als Mischungspartner unter den pannonischen Bedingungen ermittelt. Die Witterungsverhältnisse waren in den drei Versuchsjahren sehr unterschiedlich und beeinflussten die Ertragsdaten der Mischkulturen. Unter den günstigen Witterungsbedingungen im ersten Versuchsjahr zeigten die Mischungen von Leindotter-Linse und Leindotter-Platterbse ertragliche Vorteile. Im Mittel über die drei Versuchsjahre war eine Leindotter-Linsen Mischung von 25:75 Prozentanteilen die Mischung mit dem günstigsten Mischungseffekt. Leindotter in den Mischkulturen erzielte durchschnittlich 6 dt ha–1 in den Mischungen mit Linsen sowie in den Mischungen mit Platt- und Futtererbse bei hohem Leindotteranteil. In Summe war der positive Effekt der substitutiven Lein­dotter-Linse und Leindotter-Platterbse Mischungen in den günstigen, feuchten Jahren größer als der negative Effekt im ungünstigen, trockenen Jahr. Diese Leindotter-Körnerleguminosen Mischungen werden deshalb sowohl für das Trockengebiet Ostösterreichs als auch für klimatisch ähnliche Gebiete in Ungarn oder Mitteldeutschland empfohlen.The grain legumes lens (Lens culinaris), grass pea (Lathyrus sativus) and field pea (Pisum sativum) were examined as pure crops and in different mixing ratios with false flax (Camelina sativa) on organically cultivated fields of the University of Natural Resources and Life Sciences, Vienna, in the Pannonian region in three consecutive vegetation periods. The aim of this study was to test whether a mixed stand of grain legume and false flax shows advantages compared to the pure crops despite an increased competition between the partners in the mixture under the dry conditions. Therefore, the optimum seed density for the partners in the mixed stand under the Pannonian conditions was identified. The weather conditions varied widely within the three vegetation periods and influenced the yield of the mixtures. Under the favourable weather conditions in the first experimental year, the mixed stands of lens-false flax and grass pea-false flax showed benefits in yield. On average across three very different years, a mixture of false flax : lens at 25:75 percent had the most positive mixture cropping effect. An average of 0.6 t ha–1 false flax was achieved in mixed stands with lentils and in mixtures with grass pea and field pea with high false flax percentage. In general, the positive effect of the substitutive lens-false flax and grass pea-false flax mixed stands was higher in the favourable, wet years than the negative effect in the unfavourable, dry year. We therefore recommend these grain legume-false flax mixed stands for the dry regions in Eastern Austria and for climatically similar regions in Hungary or Central Germany

Respiratory chain complex III deficiency due to mutated BCS1L : a novel phenotype with encephalomyopathy, partially phenocopied in a Bcs1l mutant mouse model

Background: Mitochondrial diseases due to defective respiratory chain complex III (CIII) are relatively uncommon. The assembly of the eleven-subunit CIII is completed by the insertion of the Rieske iron-sulfur protein, a process for which BCS1L protein is indispensable. Mutations in the BCS1L gene constitute the most common diagnosed cause of CIII deficiency, and the phenotypic spectrum arising from mutations in this gene is wide. Results: A case of CIII deficiency was investigated in depth to assess respiratory chain function and assembly, and brain, skeletal muscle and liver histology. Exome sequencing was performed to search for the causative mutation(s). The patient's platelets and muscle mitochondria showed respiration defects and defective assembly of CIII was detected in fibroblast mitochondria. The patient was compound heterozygous for two novel mutations in BCS1L, c.306A > T and c.399delA. In the cerebral cortex a specific pattern of astrogliosis and widespread loss of microglia was observed. Further analysis showed loss of Kupffer cells in the liver. These changes were not found in infants suffering from GRACILE syndrome, the most severe BCS1L-related disorder causing early postnatal mortality, but were partially corroborated in a knock-in mouse model of BCS1L deficiency. Conclusions: We describe two novel compound heterozygous mutations in BCS1L causing CIII deficiency. The pathogenicity of one of the mutations was unexpected and points to the importance of combining next generation sequencing with a biochemical approach when investigating these patients. We further show novel manifestations in brain, skeletal muscle and liver, including abnormality in specialized resident macrophages (microglia and Kupffer cells). These novel phenotypes forward our understanding of CIII deficiencies caused by BCS1L mutations.Peer reviewe

Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology

C6orf203 is an RNA-binding protein involved in mitochondrial protein synthesis.

In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain-an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process

Maintenance of respiratory chain function in mouse hearts with severely impaired mtDNA transcription

The basal mitochondrial transcription machinery is essential for biogenesis of the respiratory chain and consists of mitochondrial RNA polymerase, mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2. This triad of proteins is sufficient and necessary for mtDNA transcription initiation. Abolished mtDNA transcription caused by tissue-specific knockout of TFAM in the mouse heart leads to early onset of a severe mitochondrial cardiomyopathy with lethality within the first post-natal weeks. Here, we describe a mouse model expressing human TFAM instead of the endogenous mouse TFAM in heart. These rescue mice have severe reduction in mtDNA transcription initiation, but, surprisingly, are healthy at the age of 52 weeks with near-normal steady-state levels of transcripts. In addition, we demonstrate that heavy-strand mtDNA transcription normally terminates at the termination-associated sequence in the control region. This termination is abolished in rescue animals resulting in heavy (H)-strand transcription of the entire control region. In conclusion, we demonstrate here the existence of an unexpected mtDNA transcript stabilization mechanism that almost completely compensates for the severely reduced transcription initiation in rescue hearts. Future elucidation of the underlying molecular mechanism may provide a novel pathway to treat mitochondrial dysfunction in human pathology

Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy

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ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing.

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3\u27 phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3\u27 phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2

Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O-2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T ( p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O-2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.Peer reviewe