Reactive Oxygen Species Modulate GFAP and Vimentin Expression and Co-localization
Abstract number :
3.011
Submission category :
1. Basic Mechanisms / 1A. Epileptogenesis of acquired epilepsies
Year :
2024
Submission ID :
135
Source :
www.aesnet.org
Presentation date :
12/9/2024 12:00:00 AM
Published date :
Authors :
Presenting Author: Paola Garcia Gonzalez, BS – University of Colorado, Anschutz Medical Campus
Ariana Crary, 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: Following injury to the central nervous system (CNS), astrocytes undergo a process known as astrogliosis, which is characterized by thickened processes and increased intermediate filament (IF) protein expression. Glial Fibrillary Acidic Protein (GFAP) is a well-established marker of astrogliosis, however, the underlying mechanisms driving its upregulation remain unclear. Past work in our laboratory found increased GFAP and vimentin expression in a mouse model with neuronal specific knockout of antioxidant enzyme, superoxide dismutase (SOD2). This led us to hypothesize that reactive oxygen species (ROS) may contribute to the upregulation of GFAP and vimentin, and subsequent astrogliosis.
Methods: To explore the role of ROS in the development of astrogliosis in vitro, we asked if oxidative stress and mitochondrial-specific oxidative stress (mitoROS) was sufficient to upregulate GFAP and vimentin in primary cortical co-cultures established from Sprague Dawley E18 fetal pups. Neuronal-glial co-cultures were incubated with redox cycling agents; paraquat (PQ) and mitoparaquat (MPQ), a derivative of PQ which is mitochondrial-specific. Cells were treated at DIV14 with PQ for 48 hours and at DIV7 with MPQ for 7 days. Additionally, cultures were exposed to hypoxic (5% O2) conditions to assess if limited oxygen availability and decreased ROS production influenced GFAP and Vimentin expression. We also used immunocytochemistry to observe the morphology of our astrocytes and visualize any overlap between GFAP and vimentin in treated vs control samples.
Results: Neuronal-glial cultures treated with PQ in normoxia (18% O2) had a significant upregulation of both GFAP (p = < 0.0001) and Vimentin (p = 0.0003) mRNA compared to vehicle. Similarly, western blot data demonstrated significant upregulation in GFAP (p = < 0.0001) as well as Vimentin (p = 0.0002) protein expression. By contrast, cultures treated with PQ in hypoxia showed no significant changes in mRNA (p = > 0.9999) or protein expression (p = 0.3013 for GFAP; p = 0.4195 for Vimentin) compared to vehicle. Cells treated for 7 days with MPQ in normoxia showed a significant upregulation of both GFAP (p = 0.0003) and Vimentin (p = 0.0006) protein expression compared to vehicle; whereas there was no significant change in GFAP (p = 0.1741) or vimentin (p = 0.5355) protein expression in MPQ-treated cultures in hypoxia. Furthermore, immunocytochemistry data showed a clear change in astrocyte morphology after treatment with redox cycler compounds, as well an increase in vimentin and GFAP colocalization, pointing to both IF having a significant role in the development of astrogliosis.
Conclusions: These data support the hypothesis that ROS can upregulate GFAP and vimentin expression. Co-localization of GFAP and vimentin was also observed following oxidative stress, which is known to be associated with astrogliosis. Together, these findings suggest a role for ROS and mitoROS GFAP and vimentin upregulation and astrogliosis.
Funding: This work was funded by NIH Grant R37NS039587 and NIH Diversity Supplement R37NS039587-20S1
Basic Mechanisms