On the role of superoxide dismutase and protein phosphatase type-2C beta in neuronal cell death

Das Ziel dieser Arbeit war es, die Rolle von Enzymen, den endogenen Sauerstoffradikalfängern Superoxiddismutase 1 und 2 (SOD1/2) sowie der Proteinphosphatase Typ 2C (PP2C), bei verschieden Modellen von neuronalem Zelltod zu beleuchten, um das Verständnis der zellulären Vorgänge nach neuronaler Schädigung, wie Schlaganfall, Trauma oder neurodegenerativen Erkrankungen, wie Morbus Huntington, zu verbessern. Zwei verschiedene in vivo-Modelle, das Kälte-induzierte Hirntrauma (Cold injury-induced brain trauma, CIBT) und die intrastriatale Injektion des Toxins 3-Nitropropionsäure (3-NP), fanden Anwendung in Mäusen, um den Einfluss der Menge an SOD auf das Ausmaß von Hirnschädigung zu untersuchen. Die mögliche Rolle der PP2C bei der Induktion der Apoptose, eines Enzyms, welches an der reversiblen Phosphorylierung von Proteinen beteiligt ist, wurde an kultivierten Rattenneuronen beleuchtet. Im ersten Teil dieser Arbeit wurde demonstriert, dass das zytosolische Enzym Cu/Zn- Superoxiddismutase (SOD1) den Verlauf der Schädigung nach intrastriataler Injektion von 3-NP beeinflusst. Vermehrte Expression von SOD1 in transgenen Mäusen wirkte in diesem überwiegend nekrotischen Schädigungsmodell neuroprotektiv, verkleinerte die Läsion und verminderte Ödembildung, während eine Verminderung dieses Enzyms in „knock-out“- Mutanten allein die Ödembildung signifikant verstärkte. Die Ergebnisse dieser Studie untermauern die Hypothese, dass auch im Modell der intrastriatalen Injektion die Bildung von freien Sauerstoffradikalen ein wichtiger Faktor der Intoxikation durch 3-NP ist. Dieses Model erlaubt durch Erkenntnisse über den Zelluntergang durch Energiemangel Einblicke in neurodegenerative Erkrankungen wie Chorea Huntington und erleichtert so die Suche nach therapeutischen Zielen zur Behandlung dieser Erkrankungen. Weiterhin wurde in dieser Arbeit die Rolle der mitochondrialen Mn-Superoxiddismutase (SOD2) anhand eines Modells von Hirntrauma durch Kälteschädigung (CIBT) untersucht. Die verminderte Expression von SOD2 in “knock-out” Mutanten beeinflusste den Schädigungsverlauf nach CIBT nicht. Dagegen wurde ein Anstieg der Produktion von freien Sauerstoffradikalen in der Läsion detektiert, welches Funde aus früheren Publikationen unterstützt, die postulieren, dass die Bildung reaktiver Sauerstoffspezies eine wichtige Rolle in der Schädigung nach CIBT spielt. Da SOD2 in den Mitochondrien lokalisiert ist, erlaubt dieses Ergebnis den Rückschluss, dass entweder die Produktion von freien Radikalen nicht mit der SOD2 in den Mitochondrien kolokalisiert war, wobei SOD2 keinen Einfluss auf den Spiegel freier Sauerstoffradikale haben konnte, oder dass die Menge an freigesetzten Sauerstoffradikalen in Wildtyptieren schon zu schädigend war, um eine Verschlimmerung der Läsion nach Verringerung von SOD2 zuzulassen. Außerdem zeigte diese Studie durch Verfolgen der Ödementwicklung und den Befund von haemorrhagischen Veränderungen und Entzündungsreaktionen, dass CIBT ein reproduzierbares Modell für einen biphasischen Bluthirnschranken-Zusammenbruch ist. Biphasischer Zusammenbruch der Bluthirnschranke und Entzündungsreaktionen sind Faktoren, welche bei Schlaganfall oder Hirntrauma eine Rolle spielen. Das an diesen Modellen erarbeitete Wissen kann dazu beitragen, neue Therapien zu entwickeln, um die Prognose der Patienten zu verbessern. Die Untersuchung der Rolle der PP2C in der neuronalen Apoptose war der dritte Forschungsschwerpunkt dieser Arbeit. Proteinkinasen und Phosphatasen, darunter PP2C, üben durch reversible Proteinphosphorylierung eine entscheidende regulierende Funktion in vielen zellulären Mechanismen aus. Die Ergebnisse dieser Arbeit unterstreichen die Hypothese, daß PP2C am programmierten neuronalen Zelltod (Apoptose) beteiligt sein könnte. Es wurde gezeigt, dass Aktivatoren der PP2C, wie Ölsäure oder Ginkgolsäuren, in kultivierten Rattenneuronen zeit- und konzentrationsabhängig Apoptose hervorrufen. Dagegen zeigte sich bei Verwendung des Enantiomers trans-Ölsäure (Elaidinsäure), welche PP2C in-vitro nicht aktiviert, keine Induktion von Apoptose. Ein möglicher apoptotischer Mechanismus, die Induktion durch Dephosphorylierung des proapoptotischen Proteins Bad mittels aktivierter PP2C, wurde durch Bestimmung der Menge an Ser155-phosphoryliertem Bad sowie von Gesamt-Bad (phosphoryliert + unphosphoryliert) in kultivierten Rattenneuronen beleuchtet, welche mit Ölsäure oder Ginkgolsäuren behandelt wurden. Veränderungen von unphosphoryliertem Bad konnten durch die hier eingesetzte Westernblot-Methode nicht bestimmt werden, denn für den Nachweis von unphosphoryliertem Bad standen keine Antikörper zu Verfügung. Die gemessenen Phospho-Bad/Gesamt-Bad-Veränderungen in behandelten Zellen liessen ohne die Kenntnis über das Verhältnis von Gesamt-Bad zu phosphoryliertem Bad in unbehandelten Zellen ebenfalls keinen indirekten Rückschluss auf unphosphoryliertes Bad zu

MALDI-TOF High Mass Calibration up to 200 kDa Using Human Recombinant 16 kDa Protein Histidine Phosphatase Aggregates

Background: Protein histidine phosphatase (PHP) is an enzyme which removes phosphate groups from histidine residues. It was described for vertebrates in the year 2002. The recombinant human 16 kDa protein forms multimeric complexes in physiological buffer and in the gas phase. High-mass calibration in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has remained a problem due to the lack of suitable standards. Large proteins can hardly be freed of their substructural microheterogeneity by classical purification procedures so that their use as calibrants is limited. A small adduct-forming protein of validated quality is a valuable alternative for that purpose. Methodology/Principal Findings: Three major PHP clusters of ,113, 209 and .600 kDa were observed in gel filtration analysis. Re-chromatography of the monomer peak showed the same cluster distribution. The tendency to associate was detected also in MALDI-TOF MS measuring regular adducts up to 200 kDa. Conclusions/Significance: PHP forms multimers consisting of up to more than 35 protein molecules. In MALDI-TOF MS it generates adduct ions every 16 kDa. The protein can be produced with high quality so that its use as calibration compound for high mass ranges above 100 kDa, where standards are difficult to obtain, is feasible

Loss of transforming growth factor-beta 2 leads to impairment of central synapse function

<p>Abstract</p> <p>Background</p> <p>The formation of functional synapses is a crucial event in neuronal network formation, and with regard to regulation of breathing it is essential for life. Members of the transforming growth factor-beta (TGF-β) superfamily act as intercellular signaling molecules during synaptogenesis of the neuromuscular junction of <it>Drosophila </it>and are involved in synaptic function of sensory neurons of <it>Aplysia</it>.</p> <p>Results</p> <p>Here we show that while TGF-β2 is not crucial for the morphology and function of the neuromuscular junction of the diaphragm muscle of mice, it is essential for proper synaptic function in the pre-Bötzinger complex, a central rhythm organizer located in the brainstem. Genetic deletion of TGF-β2 in mice strongly impaired both GABA/glycinergic and glutamatergic synaptic transmission in the pre-Bötzinger complex area, while numbers and morphology of central synapses of knock-out animals were indistinguishable from their wild-type littermates at embryonic day 18.5.</p> <p>Conclusion</p> <p>The results demonstrate that TGF-β2 influences synaptic function, rather than synaptogenesis, specifically at central synapses. The functional alterations in the respiratory center of the brain are probably the underlying cause of the perinatal death of the TGF-β2 knock-out mice.</p

Loss of transforming growth factor-beta 2 leads to impairment of central synapse function

<p>Abstract</p> <p>Background</p> <p>The formation of functional synapses is a crucial event in neuronal network formation, and with regard to regulation of breathing it is essential for life. Members of the transforming growth factor-beta (TGF-β) superfamily act as intercellular signaling molecules during synaptogenesis of the neuromuscular junction of <it>Drosophila </it>and are involved in synaptic function of sensory neurons of <it>Aplysia</it>.</p> <p>Results</p> <p>Here we show that while TGF-β2 is not crucial for the morphology and function of the neuromuscular junction of the diaphragm muscle of mice, it is essential for proper synaptic function in the pre-Bötzinger complex, a central rhythm organizer located in the brainstem. Genetic deletion of TGF-β2 in mice strongly impaired both GABA/glycinergic and glutamatergic synaptic transmission in the pre-Bötzinger complex area, while numbers and morphology of central synapses of knock-out animals were indistinguishable from their wild-type littermates at embryonic day 18.5.</p> <p>Conclusion</p> <p>The results demonstrate that TGF-β2 influences synaptic function, rather than synaptogenesis, specifically at central synapses. The functional alterations in the respiratory center of the brain are probably the underlying cause of the perinatal death of the TGF-β2 knock-out mice.</p

Regulation of GDF-15, a distant TGF-β superfamily member, in a mouse model of cerebral ischemia

GDF-15 is a novel distant member of the TGF-β superfamily and is widely distributed in the brain and peripheral nervous system. We have previously reported that GDF-15 is a potent neurotrophic factor for lesioned dopaminergic neurons in the substantia nigra, and that GDF-15-deficient mice show progressive postnatal losses of motor and sensory neurons. We have now investigated the regulation of GDF-15 mRNA and immunoreactivity in the murine hippocampal formation and selected cortical areas following an ischemic lesion by occlusion of the middle cerebral artery (MCAO). MCAO prominently upregulates GDF-15 mRNA in the hippocampus and parietal cortex at 3 h and 24 h after lesion. GDF-15 immunoreactivity, which is hardly detectable in the unlesioned brain, is drastically upregulated in neurons identified by double-staining with NeuN. NeuN staining reveals that most, if not all, neurons in the granular layer of the dentate gyrus and pyramidal layers of the cornu ammonis become GDF-15-immunoreactive. Moderate induction of GDF-15 immunoreactivity has been observed in a small number of microglial cells identified by labeling with tomato lectin, whereas astroglial cells remain GDF-15-negative after MCAO. Comparative analysis of the size of the infarcted area after MCAO in GDF-15 wild-type and knockout mice has failed to reveal significant differences. Together, our data substantiate the notion that GDF-15 is prominently upregulated in the lesioned brain and might be involved in orchestrating post-lesional responses other than the trophic support of neurons

TrkB and neurotrophin-4 are important for development and maintenance of sympathetic preganglionic neurons innervating the adrenal medulla

The adrenal medulla receives its major presynaptic input from sympathetic preganglionic neurons that are located in the intermediolateral (IML) column of the thoracic spinal cord. The neurotrophic factor concept would predict that these IML neurons receive trophic support from chromaffin cells in the adrenal medulla. We show here that adrenal chromaffin cells in the adult rat store neurotrophin (NT)-4, but do not synthesize or store detectable levels of BDNF or NT-3, respectively. Preganglionic neurons to the adrenal medulla identified by retrograde tracing with fast blue or Fluoro-Gold (FG) express TrkB mRNA. After unilateral destruction of the adrenal medulla, 24% of IML neurons, i.e., all neurons that are preganglionic to the adrenal medulla in spinal cord segments T7-T10, disappear. Administration of NT-4 in gelfoams (6 microgram) implanted into the medullectomized adrenal gland rescued all preganglionic neurons as evidenced by their presence after 4 weeks. NT-3 and cytochrome C were not effective. The action of NT-4 is accompanied by massive sprouting of axons in the vicinity of the NT-4 source as monitored by staining for acetylcholinesterase and synaptophysin immunoreactivity, suggesting that NT-4 may enlarge the terminal field of preganglionic nerves and enhance their access to trophic factors. Analysis of TrkB-deficient mice revealed degenerative changes in axon terminals on chromaffin cells. Furthermore, numbers of FG-labeled IML neurons in spinal cord segments T7-T10 of NT-4-deficient adult mice were significantly reduced. These data are consistent with the notion that NT-4 from chromaffin cells operates through TrkB receptors to regulate development and maintenance of the preganglionic innervation of the adrenal medulla

L-arginine Supplementation Improves Responses to Injury and Inflammation in Dextran Sulfate Sodium Colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y+ cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS−/− mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity

Equivalence of Conventionally-Derived and Parthenote-Derived Human Embryonic Stem Cells

As human embryonic stem cell (hESC) lines can be derived via multiple means, it is important to determine particular characteristics of individual lines that may dictate the applications to which they are best suited. The objective of this work was to determine points of equivalence and differences between conventionally-derived hESC and parthenote-derived hESC lines (phESC) in the undifferentiated state and during neural differentiation.hESC and phESC were exposed to the same expansion conditions and subsequent neural and retinal pigmented epithelium (RPE) differentiation protocols. Growth rates and gross morphology were recorded during expansion. RTPCR for developmentally relevant genes and global DNA methylation profiling were used to compare gene expression and epigenetic characteristics. Parthenote lines proliferated more slowly than conventional hESC lines and yielded lower quantities of less mature differentiated cells in a neural progenitor cell (NPC) differentiation protocol. However, the cell lines performed similarly in a RPE differentiation protocol. The DNA methylation analysis showed similar general profiles, but the two cell types differed in methylation of imprinted genes. There were no major differences in gene expression between the lines before differentiation, but when differentiated into NPCs, the two cell types differed in expression of extracellular matrix (ECM) genes.These data show that hESC and phESC are similar in the undifferentiated state, and both cell types are capable of differentiation along neural lineages. The differences between the cell types, in proliferation and extent of differentiation, may be linked, in part, to the observed differences in ECM synthesis and methylation of imprinted genes