Erythropoietin protects the developing brain from hyperoxia-induced cell death and proteome changes.

OBJECTIVE: Oxygen toxicity has been identified as a risk factor for adverse neurological outcome in survivors of preterm birth. In infant rodent brains, hyperoxia induces disseminated apoptotic neurodegeneration. Because a tissue-protective effect has been observed for recombinant erythropoietin (rEpo), widely used in neonatal medicine for its hematopoietic effect, we examined the effect of rEpo on hyperoxia-induced brain damage. METHODS: Six-day-old C57Bl/6 mice or Wistar rats were exposed to hyperoxia (80% O(2)) or normoxia for 24 hours and received rEpo or normal saline injections intraperitoneally. The amount of degenerating cells in their brains was determined by DeOlmos cupric silver staining. Changes of their brain proteome were determined through two-dimensional electrophoresis and mass spectrometry. Western blot, enzyme activity assays and real-time polymerase chain reaction were performed for further analysis of candidate proteins. RESULTS: Systemic treatment with 20,000 IE/kg rEpo significantly reduced hyperoxia-induced apoptosis and caspase-2, -3, and -8 activity in the brains of infant rodents. In parallel, rEpo inhibited most brain proteome changes observed in infant mice when hyperoxia was applied exclusively. Furthermore, brain proteome changes after a single systemic rEpo treatment point toward a number of mechanisms through which rEpo may generate its protective effect against oxygen toxicity. These include reduction of oxidative stress and restoration of hyperoxia-induced increased levels of proapoptotic factors, as well as decreased levels of neurotrophins. INTERPRETATION: These findings are highly relevant from a clinical perspective because oxygen administration to neonates is often inevitable, and rEpo may serve as an adjunctive neuroprotective therapy.

Brief alteration of NMDA or GABAA receptor-mediated neurotransmission has long term effects on the developing cerebral cortex.

Neurotransmitter signaling is essential for physiologic brain development. Sedative and anticonvulsant agents that reduce neuronal excitability via antagonism at N-methyl-D-aspartate receptors (NMDARs) and/or agonism at gamma-aminobutyric acid subtype A receptors (GABA(A)Rs) are applied frequently in obstetric and pediatric medicine. We demonstrated that a 1-day treatment of infant mice at postnatal day 6 (P6) with the NMDAR antagonist dizocilpine or the GABA(A)R agonist phenobarbital not only has acute but also long term effects on the cerebral cortex. Changes of the cerebral cortex proteome 1 day (P7), 1 week (P14), and 4 weeks (P35) following treatment at P6 suggest that a suppression of synaptic neurotransmission during brain development dysregulates proteins associated with apoptosis, oxidative stress, inflammation, cell proliferation, and neuronal circuit formation. These effects appear to be age-dependent as most protein changes did not occur in mice subjected to such pharmacological treatment in adulthood. Previously performed histological evaluations of the brains revealed widespread apoptosis and decreased cell proliferation following such a drug treatment in infancy and are thus consistent with brain protein changes reported in this study. Our results point toward several pathways modulated by a reduction of neuronal excitability that might interfere with critical developmental events and thus affirm concerns about the impact of NMDAR- and/or GABA(A)R-modulating drugs on human brain development.

Study of posttranslational modifications in lenticular alphaA-Crystallin of mice using proteomic analysis techniques.

In the present work the complexity in the 2D-gel protein pattern of murin lenticular alphaA-Crystallin was analyzed. An in depth study of the different protein isoforms was done combining different proteomic tools. Lens proteins of four different ages, from embryo to 100-week-old mice, were separated by large 2D-PAGE, revealing an increase in the number and intensity of the spots of alphaA-Crystallin during the process of aging. For further analyses the oldest mice were chosen. Comparison and evaluation of two different staining methods proved Imidazole-Zinc to be a good alternative to the generally used Coomassie stain. The characterization of the different alphaA-Crystallin protein species was done using nanoLC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry). Data interpretation was done by database searching, manual validation and a new MS/MS-interpretation tool for posttranslational modifications--the PTM-Explorer. Using this way, eight different phosphorylation sites were identified and localized; the identification of four of them was not published so far. Furthermore, quantitative N-terminal acetylation of alphaA-Crystallin and variable C-terminal truncation was observed, also not published in this extent yet. The results of the mass spectrometric analysis were validated by immunoblotting experiments using two different alphaA-Crystallin specific antibodies. In addition, a fluorescent phospho-specific stain was used to detect the protein spots including phosphorylation groups. Re-separation 2D-PAGE was done to round off the present study and explain the appearance of some of the protein spots in the gel as artifacts of the 2D-PAGE separation.