title: AMPA-Rezeptor und Activin A im Kontext neuronaler Exzitotoxizität im unreifen ZNS der Maus – Vergleich von Wildtyp und transgener Maus (dnActRIB)
Creators: name: Zoglauer, Daniel
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2024

description: Background: Neonatal brain hypoxic-ischemic injury represents a serious health care and socio-economical problem since it is one of the most common causes of mortality and morbidity of newborns. Severity of hypoxic-ischemic encephalopathy, with an incidence of about 2-4 per 1,000 live births and mortality rate up to 20%, varies depending on the intensity and duration of hypoxia-ischemia, on the type and size of the brain region affected, and on the maturity of the neonatal brain resulting in more or less pronounced cognitive and behavioral deficits, such as seizures, cerebral palsy or behavioural and learning disabilities. Age-dependent glutamate excitotoxicity triggered by hypoxia and/or ischemia plays a central role during the vulnerable phase of perinatal brain growth resulting in different detrimental cascades including neuroinflammation, intracellular Ca(2+) overload, production of oxygen-free radicals and apoptosis and/or necrosis. Existing neuroprotective strategies so far are little specific and insufficient in many cases. Objectives: Based on an established neonatal mouse model of acute systemic hypoxia and a mouse model of pilocarpine-induced seizures the present study investigated the age dependent regulation of AMPA receptor expression in immature CNS by using real-time PCR. Two mouse strains were compared under hypoxic conditions (8% O2, 6h), the C57Bl/6 wildtype and a C57Bl/6 transgenic mouse expressing a dominant-negative mutant of activin receptor 1B (dnActRIB) in forebrain neurons. The intention was to evaluate a hypothetical link between AMPA receptor mediated excitotoxicity and previously demonstrated neuroprotective effects of activin A. Additional Adar, an RNA-specific adenosine deaminase and AMPA receptor editing enzyme, and calcineurin, a neuronal expressed, Ca2+ -dependent phosphatase and second messenger of glutamatergic stimulation, were analyzed in neonatal mouse brain with regard to developmentally regulated mRNA expression under excitotoxic conditions. Methods: Real time polymerase chain reaction (TaqMan-rt-PCR) was used to investigate cerebral mRNA expression of AMPA receptor subunits 1-4 (GluR1-4), Adar1-3 and calcineurin at different developmental stages (postnatal day P0, P1, P3, P7, P10, P14, P17, P21 and P60). Furthermore, cerebral mRNA expression of AMPA receptor subunits 1-4 (GluR1-4), Adar1-3 and calcineurin was analyzed under hypoxic conditions, treating C57Bl/6 wildtype mice (P10) and transgenic mice (dnActRIB, P10) with 8% O2, 6h. Additionally, mRNA expression of AMPA receptor, Adar1-3 and calcineurin in CNS of C57Bl/6 wildtype mice (P10) was investigated after pilocarpine-induced seizures using rt-PCR. Results: While all AMPA subunits were already expressed in mouse brains at P0, only GluR1 and GluR3 mRNA increased significantly until adulthood (P60). mRNA levels of Adar1 and Adar2 showed an age-dependent increase, whereas Adar3 mRNA remained at a constant level of expression. After pilocarpine-induced seizures, GluR2 mRNA expression dropped significantly, whereas mRNA of GluR1 and GluR4 was unchanged 24h after seizure activity. In response to seizures Adar2 mRNA levels significantly decreased early at 1h after seizure induction. Calcineurin B1 showed no significant changes of mRNA expression in this pilocarpine-induced seizure model. After exposure to 8% O2 6h, GluR4 mRNA levels decreased significantly in wildtype, whereas mRNA concentration remained unchanged in transgenic mice due to hypoxia. In the present study hypoxia had no effect on mRNA expression of GluR1, GluR2 and GluR3. Cerebral mRNA concentrations of calcineurin B1 and Adar1-3 in wildtype as well as in transgenic mice showed no detectable hypoxia-induced alteration. Conclusions: Ca2+ -permeable AMPA receptors, as a consequence of reduced GluR2 expression in immature brain, correlate with age-dependent susceptibility to excitotoxic brain damage. Moreover, seizure induced decrease of GluR2 and Adar2 mRNA in immature brain seem to play a role during initial phase of excitotoxic neuronal cell death. Further research is required to evaluate if the presently demonstrated seizure induced decrease of GluR2 mRNA also affects GluR2 protein levels, respectively AMPA receptor function, and if GluR2 mRNA editing is influenced by early decrease of Adar2 mRNA after pilocarpine-induced seizures. According to different GluR4 mRNA expression in response to hypoxia in wildtype and transgenic mice activin A might functionally interact with AMPA receptors in immature mouse brain. Further investigation is needed to clarify if also GluR4 protein levels decrease due to hypoxia in wildtype mice and if so, would GluR4 protein concentration remain unaffected by hypoxia in the brain of transgenic mice as observed at mRNA level? So far it remains unclear if or rather how activin A respectively activin type IB receptor interacts with GluR4 expression and in consequence with AMPA receptor function. In the present study, neither acute hypoxia nor pilocarpine-induced seizures affected mRNA expression of calcineurin B1 and this obviously was not influenced by inhibited function of activin type IB receptor. This data suggests that previously described neuroprotective effects of calcineurin seem to be regulated beyond transcription.