Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence

The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development

MTO1 mediates tissue specificity of OXPHOS defects via tRNA modification and translation optimization, which can be bypassed by dietary intervention

Mitochondrial diseases often exhibit tissue-specific pathologies, but this phenomenon is poorly understood. Here we present regulation of mitochondrial translation by the Mitochondrial Translation Optimization Factor 1, MTO1, as a novel player in this scenario. We demonstrate that MTO1 mediates tRNA modification and controls mitochondrial translation rate in a highly tissue-specific manner associated with tissue-specific OXPHOS defects. Activation of mitochondrial proteases, aberrant translation products, as well as defects in OXPHOS complex assembly observed in MTO1 deficient mice further imply that MTO1 impacts translation fidelity. In our mouse model, MTO1-related OXPHOS deficiency can be bypassed by feeding a ketogenic diet. This therapeutic intervention is independent of the MTO1-mediated tRNA modification and involves balancing of mitochondrial and cellular secondary stress responses. Our results thereby establish mammalian MTO1 as a novel factor in the tissue-specific regulation of OXPHOS and fine tuning of mitochondrial translation accurac

Long-term survival in a child with severe encephalopathy, multiple respiratory chain deficiency and GFM1 mutations

BACKGROUND: Mitochondrial diseases due to deficiencies in the mitochondrial oxidative phosphorylation system (OXPHOS) can be associated with nuclear genes involved in mitochondrial translation, causing heterogeneous early onset and often fatal phenotypes. CASE REPORT: The authors describe the clinical features and diagnostic workup of an infant who presented with an early onset severe encephalopathy, spastic-dystonic tetraparesis, failure to thrive, seizures and persistent lactic acidemia. Brain imaging revealed thinning of the corpus callosum and diffuse alteration of white matter signal. Genetic investigation confirmed two novel mutations in the GFM1 gene, encoding the mitochondrial translation elongation factor G1 (mtEFG1), resulting in combined deficiencies of OXPHOS. DISCUSSION: The patient shares multiple clinical, laboratory and radiological similarities with the 11 reported patients with mutations involving this gene, but presents with a stable clinical course without metabolic decompensations, rather than a rapidly progressive fatal course. Defects in GFM1 gene confer high susceptibility to neurologic or hepatic dysfunction and this is, to the best of our knowledge, the first described patient who has survived beyond early childhood. Reporting of such cases is essential so as to delineate the key clinical and neuroradiological features of this disease and provide a more comprehensive view of its prognosis

Foxn1 Is Dynamically Regulated in Thymic Epithelial Cells during Embryogenesis and at the Onset of Thymic Involution

Thymus function requires extensive cross-talk between developing T-cells and the thymic epithelium, which consists of cortical and medullary TEC. The transcription factor FOXN1 is the master regulator of TEC differentiation and function, and declining Foxn1 expression with age results in stereotypical thymic involution. Understanding of the dynamics of Foxn1 expression is, however, limited by a lack of single cell resolution data. We have generated a novel reporter of Foxn1 expression, Foxn1G, to monitor changes in Foxn1 expression during embryogenesis and involution. Our data reveal that early differentiation and maturation of cortical and medullary TEC coincides with precise sub-lineage-specific regulation of Foxn1 expression levels. We further show that initiation of thymic involution is associated with reduced cTEC functionality, and proportional expansion of FOXN1-negative TEC in both cortical and medullary sub-lineages. Cortex-specific down-regulation of Foxn1 between 1 and 3 months of age may therefore be a key driver of the early stages of age-related thymic involution

Keep the fire burning: Current avenues in the quest of treating mitochondrial disorders

Mitochondrial diseases are very heterogeneous in their genetic cause and clinical manifestation. During the last few decades progress has been made in the diagnosis of mitochondrial diseases, but an established therapy is so far lacking. Several experimental strategies targeting different points of intervention are currently being assessed world-wide. Numerous mouse models of OXPHOS disorders have become available enabling further optimization and validation of therapeutic strategies and paving the way for future clinical trials. In this review, we provide an update on current developments towards treatment as well as the potential and status of transition into therapeutic use. (C) 2015 Elsevier B.V. and Mitochondria Research Society. All rights reserved

Defining the action spectrum of potential PGC-1 activators on a mitochondrial and cellular level in vivo

Previous studies have demonstrated a therapeutic benefit of pharmaceutical PGC-1 activation in cellular and murine model of disorders linked to mitochondrial dysfunction. While in some cases, this effect seems to be clearly associated with boosting of mitochondrial function, additional alterations as well as tissue- and cell-type-specific effects might play an important role. We initiated a comprehensive analysis of the effects of potential PGC-1-activating drugs and pharmaceutically targeted the PPAR (bezafibrate, rosiglitazone), AMPK (AICAR, metformin) and Sirt1 (resveratrol) pathways in HeLa cells, neuronal cells and PGC-1-deficient MEFs to get insight into cell type specificity and PGC-1 dependence of their working action. We used bezafibrate as a model drug to assess the effect on a tissue-specific level in a murine model. Not all analyzed drugs activate the PGC pathway or alter mitochondrial protein levels. However, they all affect supramolecular assembly of OXPHOS complexes and OXPHOS protein stability. In addition, a clear drug- and cell-type-specific influence on several cellular stress pathways as well as on post-translational modifications could be demonstrated, which might be relevant to fully understand the action of the analyzed drugs in the disease state. Importantly, the effect on the activation of mitochondrial biogenesis and stress response program upon drug treatment is PGC-1 dependent in MEFs demonstrating not only the pleiotropic effects of this molecule but points also to the working mechanism of the analyzed drugs. The definition of the action spectrum of the different drugs forms the basis for a defect-specific compensation strategy and a future personalized therapeutic approach

Proportional expansion of <i>Foxn1</i>^<i>neg</i> cTEC and mTEC is driven by different mechanisms.

<p><b>(A,B)</b> Plots show proportion of GFP^- <b>(A)</b> and numbers of GFP⁺ and GFP^- (B) mTEC and cTEC at the ages shown, as determined by flow cytometric analysis. (<b>C</b>) Flow cytometric analysis showing proportion of active caspase-3⁺ cells in the populations shown. (<b>C’</b>) shows data in (<b>C</b>) presented to indicate the relative levels of apoptosis in GFP⁺ and GFP^- cTEC and mTEC. <b>(D)</b> Flow cytometric analysis of thymi from 6 week old mice showing proportion of Ki67⁺ cells in the populations shown. <b>(E)</b> GFP expression profile in cTEC and mTEC at the ages shown. (F) Quantification of data displayed in (E) showing median fluorescence intensity (MFI) of GFP⁺ cTEC and mTEC at 1 month and 3 months. <b>(A)</b> n = 5, (B) n = 7, (<b>C</b>) n = 3 (<b>D</b>) n = 4, (<b>E, F</b>) n = 5 independent biological experiments.</p

Proportional expansion of <i>Foxn1</i>^<i>neg</i> TEC occurs at the onset of age-related thymic involution.

<p><b>(A)</b> Thymus involution in <i>Foxn1</i>^<i>G/+</i> mice occurs with normal kinetics. <b>(C, D, E)</b> Flow cytometric analysis of <i>Foxn1</i>^<i>G/+</i> thymi at the ages shown; the proportion of GFP- TEC increases with age. Red (C, D) and grey (E) lines show FMO. (<b>B</b>) Absolute number of GFP⁺ and GFP^- TEC isolated from <i>Foxn1</i>^<i>G/+</i> thymi at the timepoints shown. Absolute numbers are as follows: 1 month; GFP⁺ 4.94x10⁴±1.81x10⁴, GFP^- 1.06x10⁴±3.07x10³. 3 months; GFP⁺ 4.03x10⁴±8.86x10³, GFP^- 1.31x10⁴±3.01 x 10³. 12 months; GFP⁺ 1.55x10⁴±2.36x10³, GFP^- 6.55x10³±1.88x10³. 24 months; GFP⁺ 4.52x10³±2.75x10³, GFP^- 1.81x10³±1.10x10³. <b>(A,B,C)</b> n = 3, (<b>D,E</b>) n = 2 independent biological experiments.</p

Dynamic regulation of <i>Foxn1</i> is evident in the early stages of TEC differentiation.

<p><b>(A-D)</b> Flow cytometric analysis of E13.5, E15.5 and E17.5 fetal <i>Foxn1</i>^<i>G/+</i> thymic primordia for the markers shown. Plots show data after gating against Lineage⁺ and on total EpCAM⁺ cells. WT, wild type. <b>(A-C)</b> n = 3, (<b>D</b>) n = 4 independent biological experiments.</p