Angela Hübner

Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Computational analysis of liquid chromatography-tandem mass spectrometric steroid profiling in NCI H295R cells following angiotensin II, forskolin and abiraterone treatment

Adrenal steroid hormones, which regulate a plethora of physiological functions, are produced via tightly controlled pathways. Investigations of these pathways, based on experimental data, can be facilitated by computational modeling for calculations of metabolic rate alterations. We therefore used a model system, based on mass balance and mass reaction equations, to kinetically evaluate adrenal steroidogenesis in human adrenal cortex-derived NCI H295R cells. For this purpose a panel of 10 steroids was measured by liquid chromatographic-tandem mass spectrometry. Time-dependent changes in cell incubate concentrations of steroids - including cortisol, aldosterone, dehydroepiandrosterone and their precursors - were measured after incubation with angiotensin II, forskolin and abiraterone. Model parameters were estimated based on experimental data using weighted least square fitting. Time-dependent angiotensin II- and forskolin-induced changes were observed for incubate concentrations of precursor steroids with peaks that preceded maximal increases in aldosterone and cortisol. Inhibition of 17-alpha-hydroxylase/17,20-lyase with abiraterone resulted in increases in upstream precursor steroids and decreases in downstream products. Derived model parameters, including rate constants of enzymatic processes, appropriately quantified observed and expected changes in metabolic pathways at multiple conversion steps. Our data demonstrate limitations of single time point measurements and the importance of assessing pathway dynamics in studies of adrenal cortical cell line steroidogenesis. Our analysis provides a framework for evaluation of steroidogenesis in adrenal cortical cell culture systems and demonstrates that computational modeling-derived estimates of kinetic parameters are an effective tool for describing perturbations in associated metabolic pathways.

Homozygous ARHGEF2 mutation causes intellectual disability and midbrain-hindbrain malformation

Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder.

Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers

Investigating cell division in human adrenal cells we show that proliferation is decreased upon overexpression of ALADIN, PGRMC1 or PGRMC2. In immunofluorescence experiments using human adrenal cells and triple A patient fibroblasts we observed that during cell division PGRMC1 localizes to the microtubule kinetochore-fibers in metaphase and to the mid-body in telophase. Depletion of ALADIN results in mis-localization of Aurora A and PGRMC1 in metaphase cells of the human adrenal cell line and fibroblasts derived from patients with triple A syndrome. In real time PCR using RNA of fibroblasts of triple A syndrome patients and healthy controls we measured an increased expression of PGRMC2 in cells with ALADIN mis-function compared to the control cells. We hypothesize that a loss of the regulatory interaction between ALADIN and PGRMC2 leads to an over-regulation and over-expression of PGRMC2 and displaces PGRMC1 at the metaphase spindle. This diminishing of PGRMC1 concentration at kinetochore fibers may lead to mitotic errors and pro - liferation arrest. ABSTRACT Membrane-associated progesterone receptors are restricted to the endoplasmic reticulum and are shown to regulate the activity of cytochrome P450 enzymes which are involved in steroidogenesis or drug detoxification. PGRMC1 and PGRMC2 belong to this group of microsomal receptors and are of interest due to their suspected role during cell cycle. PGRMC1 and PGRMC2 are thought to bind to each other thereby suppressing entry into mitosis. We could previously report that PGRMC2 interacts with the nucleoporin ALADIN which when mutated results in the autosomal recessive disorder triple A syndrome. ALADIN is a novel regulator of mitotic controller Aurora kinase A and depletion of this nucleoporin leads to microtubule instability. In the current study, we present that proliferation is decreased when ALADIN, PGRMC1 or PGRMC2 are over-expressed. Furthermore, we find that depletion of ALADIN results in mis-localization of Aurora kinase A and PGRMC1 in metaphase cells. Additionally, PGRMC2 is over-expressed in triple A patient fibroblasts. Our results emphasize the possibility that loss of the regulatory interaction between ALADIN and PGRMC2 gives rise to a depletion of PGRMC1 at kinetochore fibers and to mitotic errors. This observation may explain part of the symptoms seen in triple A syndrome patients.

Molekulare und genetische Zusammenhänge

Das Verstandnis der molekularen Grundlage endokriner Erkrankungen ist durch die Methoden der Molekularbiologie, Molekulargenetik und Zellbiologie in den letzten 25 Jahren sprunghaft angestiegen. Ist die Pathophysiologie einiger monogener Erkrankungen wie dem adrenogenitalen Syndrom weitgehend aufgeklart, so sind Krankheitsbilder wie die Adipositas oder der Diabetes mit ihrer moglicherweise polygenen oder multifaktoriellen Genese wenig verstanden. Die Anwendung molekular- und zellbiologischer Methoden in der padiatrischen Endokrinologie hat nicht nur zu einer Fulle neuer Informationen gefuhrt, sondern auch die Komplexitat der wissenschaftlichen Fragestellungen in der klinischen Endokrinologie drastisch erhoht. Das Verstandnis der grundlegenden Zusammenhange, Methoden und Nomenklaturen der Molekularbiologie ist eine wichtige Voraussetzung geworden, die atiologie und die neuen Therapieansatze endokriner Erkrankungen zu verstehen (Kopp 2005).