RhoA inhibition and Rac1 Activation Increases Mitochondrial Respiration in Propofol Induced Neonatal Neurotoxicity

Background Mitochondrial function is critical to neuronal health. Several neurodegenerative diseases such as Alzheimer's and Huntington's exhibit some form of mitochondrial dysfunction along with impaired actin dynamics. Previously, we have shown that propofol exposure in neonatal mice (PND7) leads to RhoA activation, growth cone collapse, impaired axonal transport of neurotrophic factors, and learning deficits observed three months later. RhoA inhibition prior to propofol is protective against neurotoxicity. Because actin dynamics are critical for mitochondrial fission/fusion and overall health, we hypothesized that propofol exposure to neonatal mouse neurons may also lead to mitochondrial dysfunction and that interventions to maintain homeostasis of RhoGTPase activity and actin dynamics would ameliorate propofol induced mitochondrial injury. Methods To assess changes in mitochondrial function after propofol exposure, embryonic mouse neurons were harvested and plated on an Agilent Seahorse XF Cell Culture Microplate for 6–7 days. Neurons were then exposed to propofol (3uM, 4 hrs) or DMSO vehicle control. A subset of neurons were pretreated with TAT‐C3(50ug/ml, 2 hrs) or EGF (1unit/ml, concurrent) to inhibit RhoA or activate Rac1, respectively. Oxygen consumption rate and extracellular acidification rate with a Seahorse XF96 extracellular flux analyzer was evaluated using the XF Cell Mito Stress Test Assay. Basal respiration and maximum respiration were measured and spare respiration was calculated by taking the difference between basal and maximum respiration. Results Embryonic neurons exposed to propofol did not show any difference in basal, maximum, or spare respiration compared to DMSO control. Neurons pretreated with TAT‐C3 followed by propofol exposure showed a significant increase in both maximum respiration and spare respiration. Neurons pretreated with EGF followed by propofol exposure showed a significant increase in basal, maximum, and spare respiration. Conclusion We present here the results of mitochondrial respiration changes in propofol induced neurotoxicity in neonatal mouse neurons. Although our results show no significant changes in mitochondrial respiration at clinically relevant doses of propofol, interventions that stabilize and balance actin dynamics have a significant impact on maximum and spare mitochondrial respiration when “stressed” by propofol. These data suggest that RhoA inhibition and/or Rac1 activation during propofol exposure to neonatal neurons may be protective by maintaining appropriate mitochondrial fission/fusion balance, and thus optimizing the mitochondria pool to respond to a cellular stress. Support or Funding Information NIH: R01 GM085179 (PI: Patel PM) NIH: K08 GM 124500‐01A1 (PI: Pearn ML)TAT‐C3 (RhoA inhibitor) pretreatment prior to propofol exposure increases maximum and spare respiration Neonatal neurons exposed to propofol (3uM, 4hrs) did not show any changes in basal, maximum, spare respiration compared to DMSO control. TAT‐C3 prior to propofol exposure resulted in a significantly increased maximum respiration (middle graph) and significantly increased spare respiration (right graph).Epidermal growth factor (EGF, Rac1 activator) pretreatment prior to propofol exposure increases basal, maximum and spare respiration Neonatal neurons exposed to propofol (3uM, 4hrs) did not show any changes in basal, maximum, spare respiration compared to DMSO control. Concurrent exposure of EGF along with propofol resulted in a significantly increased basal, maximum and spare respiration.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .