Abstract SNACC-68

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Anesthetic toxicity: RhoGTPases, growth cone collapse, and axonal transport

1Pearn M, 2DerMardirossian C, 1Kodama M, 3Wu C, 1Patel P, 1Head B
1Veteran Affairs Medical Center, San Diego, California, United states; 2Scripps Research Institute, La Jolla, , United states; 3UCSD, La Jolla, California, United states

Background: A wide body of evidence indicates that anesthetic exposure during synaptogenesis in the developing brain causes widespread neurodegeneration, electrophysiologic abnormalities and long term cognitive deficits. Although the mechanism by which anesthetics injure the neonatal brain is not known, GABA-A mediated excitation, NMDAR antagonism mediated excitotoxicity, aberrant cell cycle entry, mitochondrial injury and free radical mediated toxicity play a role. Work from our laboratory has demonstrated that preferential signaling of proBDNF via p75NTR leads to downstream activation of RhoA, actin dysregulation, loss of dendritic spines and loss of synapses. That inhibition of RhoA attenuates these adverse effects suggests that agents that inhibit RhoA might prevent cognitive deficits attendant with neonatal anesthetic exposure.
The means by which anesthetic induced actin dysregulation leads to neurotoxicity is not clear. Development of neuronal networks requires active axonal growth with appropriate targeting and formation of synapses. In addition, both retrograde and anterograde transport of material is critical to axonal growth. The axonal growth cone at the tip of the axon is required for growth and proper pathfinding. A dynamically regulated actin cytoskeleton is essential for normal growth cone morphology. Indeed, dysregulation of actin leads to growth cone collapse and aberrant synaptic connections. RhoGTPases, which include RhoA, Rac1 and Cdc42, are key regulators of the actin cytoskeleton. Moreover, these RhoGTPases are required for proper axonal transport of signaling endosomes.
We hypothesize that during the vulnerable period of DIV5-7, anesthetic exposure leads to increased RhoA activation, disruption of normal actin cytoskeleton dynamics, growth cone collapse, and impaired retrograde axonal transport. RhoA inhibition prior to anesthetic exposure will rescue neurons from anesthetic mediated growth cone collapse and retrograde axonal transport impairments.

Methods: Primary rat embryonic neurons (E18) were cultured for 5 days in vitro (DIV5-7) and exposed to propofol (PPF). A subset of neurons were pretreated with C3 toxin (pharmacological inhibitor of RhoA) or transfected with dominant negative RhoA constructs (genetic inhibitor of RhoA) prior to PPF exposure. Growth cones were stained for F-actin to assess growth cone area. Retrograde axonal transport was assayed by live cell imaging of quantum dot labeled BDNF (BDNF-QD).

Results: PPF exposure at DIV5-7 results in increased RhoA activation, dysregulation of actin dynamics, growth cone collapse, and impaired retrograde axonal transport of QD-BDNF; these deleterious effects are attenuated by RhoA inhibition prior to PPF exposure.

Conclusions: These results demonstrate for the first time that PPF exposure in developing neurons results in increased RhoA signaling, growth cone collapse and impaired retrograde axonal transport of QD-BDNF. Propofol mediated growth cone collapse and impaired axonal transport are attenuated by RhoA inhibition. These findings expand our knowledge of anesthetic mediated neurotoxicity in developing neurons and identify novel targets for therapeutic intervention in the event that toxicity is demonstrated in humans.



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