ABSTRACT
Objective: To investigate the role of oxidative stress in inhibiting forkhead box O1 (FOXO1) through activating cyclin-dependent kinase 5 (Cdk5) in neuronal damage. Methods: Cdk5 was coexpressed with p39 in HEK cells together with wild-type FOXO1 or a nonphosphorylatable FOXO1-S249A mutant; FOXO1 phosphorylation was immunoblotted through pS249-FOXO1 antibody which specific recognized FOXO1 Ser249 phosphorylation. Cdk5 or p39 was coexpressed in HEK cells together with FOXO1 and their interaction was explored through Co-IP. In primary cortical culture neurons, various Cdk5 activators (p25, p35, and p39) were overexpressed together with Cdk5. FOXO1 transcriptional activity was tested by luciferase assay. We detected the effect of Cdk5 inhibitor roscovitine on FOXO1 transcriptional activity in primary neurons. We compared FOXO1 subcellular localization of wild type with Cdk5 heterozygous mice cortical brain tissues through cytoplasmic and nuclear extracts. In addition, we administered oxidative stress (H2O2 and glutamate treatment) in primary neurons and examined the effect on the cdk5 activity by Western blot, FOXO1 transcriptional activity by luciferase assay, and neuronal damage through stained caspase3. Results: ① Identification of Cdk5/p39 was capable of phosphorylating the transcription factor FOXO1 at Ser249, and associated with it. ② Activated Cdk5 dramatically inhibited FOXO1 transcriptional activity. In contrast, roscovitine, Cdk5 inhibitor, significantly increased FOXO1 transcriptional activity and led to FOXO1 translocation into the nucleus. ③ FOXO1 protein was found predominately in the nucleus in the Cdk5 heterozygous mice. H2O2 or glutamate treatment promoted p35 to be cleaved to p25, and inhibited FOXO1 transcriptional activity. Importantly, this effect of oxidative stress was abolished when Cdk5 was silenced by shRNA. ⑤ Oxidative stress dramatically increased caspase3 immunostaining in primary neurons. Conclusion: Cdk5/p39 inhibits FOXO1 activity through phosphorylation and association. Moreover, oxidative stress inhibits FOXO1 activity through activating Cdk5, and leads to neuronal death.
ABSTRACT
<p><b>OBJECTIVE</b>To elucidate the genetic characterization and molecular epidemiological features of Echovirus 19 (E19) isolates collected from an outbreak associated with hand, foot and mouth disease (HFMD) in Tai'an city of Shandong Province of China from July to September, 2003.</p><p><b>METHODS</b>Thirty seven Echovirus 19 isolates were isolated from stool specimens and throat swabs collected during the outbreak, then major capsid (VP1) genomic sequence was determined, and phylogenetic tree was done based on the VP1 sequences among these 37 and other E19 viruses deposited in the Genbank. Also a representative strain named CHN-SD03-TN12 was selected for sequencing of 5'-untranslated regions (5'-UTR).</p><p><b>RESULTS</b>The identity rate was about 98.9%-100% among all these 37 E19 viruses. The genetic relationships between these 37 E19 isolates and other strains reported were also depicted. The identity rate was about 78.4%-78.9% compared with E19 reference strain Burke. The substitutions in the sequence of 5'-UTR resulted in changes in the conjectural properties of 5'-UTR of E19 viruses.</p><p><b>CONCLUSION</b>The genetic features of E19 viruses isolated during the outbreak in Shandong Province in 2003 may be associated with a genetic and antigenic drift that changes the virulence of the Shandong isolates, but the molecular changes in Shandong E19 viruses contributing to their phenotype remain to be further illuminated. However, the sequences described in this paper substantiate the changes taken place in capsid VP1 and 5'UTR regions. These substitutions may contribute to their tropism and virulence, and play a significant role in pathogenesis and clinical manifestations of the disease.</p>