Authors :
Presenting Author: Ariana Crary, – University of Colorado Anschutz Medical Campus
Paola Garcia Gonzalez, BS – University of Colorado Anschutz Medical Campus
Ruth Fulton, MS – University of Colorado Anschutz Medical Campus
Manisha Patel, PhD – University of Colorado Anschutz Medical Campus
Rationale:
A growing body of evidence implicates metabolic dysfunction, particularly mitochondrial reactive oxygen species (mtROS) in epilepsy pathophysiology. In fact, neuron-specific deletion of the mitochondrial antioxidant, superoxide dismutase 2 (SOD2), increasing neuronal mtROS, was sufficient to drive severe seizures and astrogliosis in SOD2fl/fl;NEX-cre mice (Fulton et al., 2021). However, the mechanism by which glial fibrillary acidic protein (GFAP) upregulation, a hallmark of astrogliosis, occurs in epilepsy and the underlying role of mtROS in the process is a critical gap in knowledge. Lipid droplet accumulation in astrocytes has been shown to occur in human and animal models of temporal lobe epilepsy and prevention of their formation decreases seizure frequency and duration (Chen et al., 2023). As lipid droplet buildup itself can trigger astrocyte reactivity, we hypothesize that neuronal mtROS promotes lipid droplet accumulation in astrocytes, potentially contributing to astrogliosis and seizure susceptibility. Methods:
To test if neuronal mtROS can increase lipid droplet accumulation in astrocytes, we performed immunohistochemistry and optimized BODIPY lipid droplet staining in the SOD2fl/fl;NEX-cre mouse model, in which seizures are initiated by increased neuronal mtROS. To determine that lipid droplet accumulation was a mtROS-specific effect rather than a secondary consequence of seizures in the mice, we moved to an in vitro model of mtROS by treating neuron-glia cultures with mitoparaquat (MPQ), a drug known to increase mtROS. We first validated that MPQ was sufficient to increase GFAP expression, a marker of astrogliosis, through RT-qPCR and western blotting. After this, we performed immunocytochemistry and BODIPY lipid droplet staining.
Results:
We found that there was a significant increase in the number of lipid droplets in the cortex of SOD2fl/fl;NEX-cre mice compared to control littermates and that they colocalized to the astrocyte marker, GFAP (N= 5, p < 0.01, student’s t-test). In the neuron-glia cultures, we found that MPQ treatment was sufficient to upregulate GFAP mRNA and protein expression and that hypoxic conditions attenuated this effect, demonstrating that mtROS was sufficient to drive an astrocyte response (p < 0.001). Next, we observed that MPQ-treated cells had a significant increase in astrocyte lipid droplets compared to control cells, indicating that mtROS is sufficient to drive astrocyte lipid droplet accumulation, rather than being a secondary effect from seizures (N=11, p < 0.001, Mann-Whitney test).