Inhibition of aberrant cyclin-dependent kinase 5 activity attenuates isoflurane neurotoxicity in the developing brain

Abstract

Aberrant CDK5 activity is implicated in a number of neurodegenerative disorders. Isoflurane exposure leads to neuronal apoptosis, and subsequent learning and memory defects in the developing brain. The present study was designed to examine whether and how CDK5 activity plays a role in developmental isoflurane neurotoxicity. Rat pups and hippocampal neuronal cultures were exposed to 1.5% isoflurane for 4 h. The protein and mRNA levels of CDK5, p35 and p25 were detected by western blot and QReal-Time PCR. CDK5 activity was evaluated in vitro using Histone H1 as a substrate. Roscovitine (an inhibitor of CDK5) was applied before isoflurane treatment, cleaved Caspase-3, Bcl-2, Bax, MEF2 and phospho-MEF2A-Ser-408 expressions were determined. Dominant-Negative CDK5 was transfected before isoflurane treatment. Neuronal apoptosis was evaluated by Flow cytometry (FCM) and TUNEL-staining. Cognitive functions were assessed by Morris water maze. We found that isoflurane treatment led to an aberrant CDK5 activation due to its activator p25 that was cleaved from p35 by calpain. Inhibition of CDK5 activity with Roscovitine enhanced Bcl-2, and decreased cleaved Caspase-3 and Bax expressions. In addition, isoflurane exposure resulted in a decrease of MEF2 and increase of phospho-MEF2A-Ser-408, which were rescued by Roscovitine or Dominant-Negative CDK5 transfection. Dominant-Negative CDK5 transfection also decreased the percentage of TUNEL-positive cells in isoflurane neurotoxicity. Moreover, Roscovitine remarkably alleviated the learning and memory deficits induced by postnatal isoflurane exposure. These results indicated that aberrant CDK5 activity-dependent MEF2 phosphorylation mediates developmental isoflurane neurotoxicity. Inhibition of CDK5 overactivation contributes to the relief of isoflurane neurotoxicity in the developing brain.

Keywords: Apoptosis; CDK5; Ca(2+)-activated protease; Calpain; Cyclin-dependent kinase 5; DIV; DN CDK5; Day in vitro; Dominant-Negative cyclin-dependent kinase 5; FCM; Flow cytometry; Hippocampus; Isoflurane; LSCM; Laser scanning confocal microscope; Learning and memory; MDL 28170; MEF2; MWM; Morris water maze; Myocyte enhancer factor 2; N-benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone; N-benzyloxycarbonylvalylphenylalaninal; Neurotoxicity; PND; Postnatal day; TUNEL; Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling; Z-VAD.fmk.