Impaired EIF2S3 function associated with a novel phenotype of X-linked hypopituitarism with glucose dysregulation

BACKGROUND: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome. // METHODS: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation. In situ hybridisation on human embryonic tissue, EIF2S3-knockdown studies in a human pancreatic cell line, and yeast assays on the mutated corresponding eIF2γ protein, were performed in this study. // FINDINGS: We report a novel hemizygous EIF2S3 variant, p.Pro432Ser, in the three boys (heterozygous in their mothers). EIF2S3 expression was detectable in the developing pituitary gland and pancreatic islets of Langerhans. Cells lacking EIF2S3 had increased caspase activity/cell death. Impaired protein synthesis and relaxed start codon selection stringency was observed in mutated yeast. // INTERPRETATION: Our data suggest that the p.Pro432Ser mutation impairs eIF2γ function leading to a relatively mild novel phenotype compared with previous EIF2S3 mutations. Our studies support a critical role for EIF2S3 in human hypothalamo-pituitary development and function, and glucose regulation, expanding the range of phenotypes associated with EIF2S3 mutations beyond classical MEHMO syndrome. Untreated hypoglycaemia in previous cases may have contributed to their more severe neurological impairment and seizures in association with impaired EIF2S3. // FUND: GOSH, MRF, BRC, MRC/Wellcome Trust and NIGMS funded this study

Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans

High-resolution MCP-TimePix3 imaging/timing detector for antimatter physics

We present a hybrid imaging/timing detector for force sensitive inertial measurements designed for measurements on positronium, the metastable bound state of an electron and a positron, but also suitable for applications involving other low intensity, low energy beams of neutral (antimatter)-atoms, such as antihydrogen. The performance of the prototype detector was evaluated with a tunable low energy positron beam, resulting in a spatial resolution of approximate t

A Transient Transgenic RNAi Strategy for Rapid Characterization of Gene Function during Embryonic Development

RNA interference (RNAi) is a powerful strategy for studying the phenotypic consequences of reduced gene expression levels in model systems. To develop a method for the rapid characterization of the developmental consequences of gene dysregulation, we tested the use of RNAi for “transient transgenic” knockdown of mRNA in mouse embryos. These methods included lentiviral infection as well as transposition using the Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems. This approach can be useful for phenotypic validation of putative mutant loci, as we demonstrate by confirming that knockdown of Prdm16 phenocopies the ENU-induced cleft palate (CP) mutant, csp1. This strategy is attractive as an alternative to gene targeting in embryonic stem cells, as it is simple and yields phenotypic information in a matter of weeks. Of the three methodologies tested, the PB transposon system produced high numbers of transgenic embryos with the expected phenotype, demonstrating its utility as a screening method

Astroglial Inhibition of NF-κB Does Not Ameliorate Disease Onset and Progression in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Motor neuron death in amyotrophic lateral sclerosis (ALS) is considered a “non-cell autonomous” process, with astrocytes playing a critical role in disease progression. Glial cells are activated early in transgenic mice expressing mutant SOD1, suggesting that neuroinflammation has a relevant role in the cascade of events that trigger the death of motor neurons. An inflammatory cascade including COX2 expression, secretion of cytokines and release of NO from astrocytes may descend from activation of a NF-κB-mediated pathway observed in astrocytes from ALS patients and in experimental models. We have attempted rescue of transgenic mutant SOD1 mice through the inhibition of the NF-κB pathway selectively in astrocytes. Here we show that despite efficient inhibition of this major pathway, double transgenic mice expressing the mutant SOD1G93A ubiquitously and the dominant negative form of IκBα (IκBαAA) in astrocytes under control of the GFAP promoter show no benefit in terms of onset and progression of disease. Our data indicate that motor neuron death in ALS cannot be prevented by inhibition of a single inflammatory pathway because alternative pathways are activated in the presence of a persistent toxic stimulus

To automate or not to automate: this is the question

New protocols and instrumentation significantly boost the outcome of structural biology, which has resulted in significant growth in the number of deposited Protein Data Bank structures. However, even an enormous increase of the productivity of a single step of the structure determination process may not significantly shorten the time between clone and deposition or publication. For example, in a medium size laboratory equipped with the LabDB and HKL-3000 systems, we show that automation of some (and integration of all) steps of the X-ray structure determination pathway is critical for laboratory productivity. Moreover, we show that the lag period after which the impact of a technology change is observed is longer than expected

Positronium Laser Cooling via the 1 3 S − 2 3 P Transition with a Broadband Laser Pulse

We report on laser cooling of a large fraction of positronium (Ps) in free flight by strongly saturating the 1^{3}S-2^{3}P transition with a broadband, long-pulsed 243 nm alexandrite laser. The ground state Ps cloud is produced in a magnetic and electric field-free environment. We observe two different laser-induced effects. The first effect is an increase in the number of atoms in the ground state after the time Ps has spent in the long-lived 2^{3}P states. The second effect is one-dimensional Doppler cooling of Ps, reducing the cloud's temperature from 380(20) to 170(20) K. We demonstrate a 58(9)% increase in the fraction of Ps atoms with v_{1D}<3.7×10^{4}  ms^{-1}

Gene expression in mouse thyrotrope adenoma: transcription elongation factor stimulates proliferation

Thyrotrope hyperplasia and hypertrophy are common responses to primary hypothyroidism. To understand the genetic regulation of these processes, we studied gene expression changes in the pituitaries of Cga-/- mice, which are deficient in the common alpha subunit of TSH, LH and FSH. These mice have thyrotrope hypertrophy and hyperplasia and develop thyrotrope adenoma. We report that cell proliferation is increased, but the expression of most stem cell markers is unchanged. The alpha subunit is required for secretion of the glycoprotein hormone beta subunits, and mutants exhibit elevated expression of many genes involved in the unfolded protein response, consistent with dilation and stress of the endoplasmic reticulum. Mutants have elevated expression of transcription factors that are important in thyrotrope function, such as Gata2 and Isl1, and those that stimulate proliferation, including Nupr1, E2f1 and Etv5. We characterized the expression and function of a novel, over-expressed gene, transcription elongation factor A (SII)-like 5 (Tceal5). Stable expression of Tceal5 in a pituitary progenitor cell line is sufficient to increase cell proliferation. Thus, Tceal5 may act as a proto-oncogene. This study provides a rich resource for comparing pituitary transcriptomes and an analysis of gene expression networks

Expression Of Anti-dna Immunoglobulin Transgenes In Non-autoimmune Mice

SELF-REACTIVE B cells can be regulated by either deletion or inactivation 1. These manifestations of self-tolerance have been dramatically shown in transgenic mice in which the number of self-reactive cells has been artificially expanded 2,3. We have now extended these models to ask if B-cell tolerance as described for non-disease-associated antigens also operates for the targets of autoimmunity. The target we have chosen is DNA. Anti-DNA antibodies are diagnostic of certain autoimmune syndromes in humans and are a characteristic of the murine model of systemic autoimmunity, the MRI/Ipr mouse4. Antibodies to both single-stranded and double-stranded DNA have been implicated in disease 5,6. By generating anti-DNA transgenic mice, we have addressed the question of whether DNA-specific B cells are regulated in normal (non-autoimmune) mice. We indeed found that most transgenic B cells bind DNA, yet we failed to detect secreted anti-DNA. We suggest that as a consequence of their self-reactivity these B cells are developmentally arrested.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62839/1/349331a0.pd