Abstracts

Rationale: Spontaneous unprovoked seizures (i.e., epilepsy) can be acquired as a result of an insult to the brain (status epilepticus; SE, trauma, infection, stroke or tumor) or genetic factors. Epileptogenic injury sets into motion many cellular and molecular events including neuroinflammation, redox alterations and mitochondrial dysfunction that may contribute to the pathogenesis of epilepsy. Increased expression of the intermediary filament glial acidic fibrillary protein (GFAP) in activated astrocytes is commonly observed in epilepsy, however, the precise mechanisms by which it is upregulated remain unclear. In this study, we determined the role of mitochondrial reactive oxygen species (mtROS) in the induction of GFAP mRNA and protein upregulation associated with epilepsy.

Methods: We selectively deleted the most prominent mitochondrial antioxidant, manganese superoxide dismutase-2 (SOD2) in neuronal cells expressing the basic helix-loop-helix transcription factor (NEX) through the cre-lox system. This restricts deletion to a subset of principal neurons primarily in the forebrain. SOD2^fl/fl NEX^Cre/Cre (henceforth SOD2^-/- mice), and wild-type mice were analyzed for seizures using video-EEG analysis and further probed for altered expression of GFAP mRNA and protein by RTPCR and Western blot analysis. Parallel experiments were performed in primary neuron-glial or astrocyte-isolated rat cerebro-cortical cultures treated with a mtROS generating compound. Neuronal hyperexcitability was assessed by a multiple electrode array system.

Results: Previous transcriptome-wide gene expression profiling of forebrain from SOD2^-/- mice vs their WT littermates revealed the highest fold-change in GFAP mRNA followed by vimentin; this result was confirmed via reverse transcription PCR (RTPCR) (4.4327 fold increase, p=5.30 X 10-17). This suggests that mitochondrial ROS originating from neurons in SOD2^-/- mice is sufficient to upregulate GFAP mRNA in astrocytes. To further address this mechanistically, we elevated mitochondrial ROS in primary cultures containing mixed neurons and glia with a mitochondrial-specific redox cycling agent, mito-paraquat (mitoPQ). Primary neuronal-glial cultures treated for 1 week with mitoPQ (0, 1 and 5uM) showed a concentration-dependent increase in GFAP mRNA, replicating one aspect of the in-vivo model. MitoPQ induced GFAP mRNA induction was prevented in astrocyte-rich cultures or when grown in under hypoxic conditions, suggesting the role of neurons and ROS respectively. Western blot analysis of GFAP protein in SOD2^-/- mice (n=4 mice per condition) and mito-PQ treated cultures (n=3 biological replicates per condition) showed increased fragmentation compared to vehicle treated controls suggesting oxidative degradation.

Conclusions: Collectively, these data indicate that neuronal mtROS is sufficient to induce an increase in astrocytic GFAP mRNA and protein levels. Furthermore, oxidative posttranslational modifications of GFAP could contribute to the upregulation and degradation of GFAP following epileptogenic brain injury.

Funding: Please list any funding that was received in support of this abstract.: Supported by grants R01NS039587, RO1NS039587S1, R01 NS086423 and R01NS086423S1 from NINDS and the ALSAM Therapeutic Innovation Award (M.P.).