Kainic Acid Induces p53 Accumulation and Neuronal Apoptosis Through Downregulation of Ubiquitin
Abstract number :
1.049
Submission category :
Year :
2000
Submission ID :
1421
Source :
www.aesnet.org
Presentation date :
12/2/2000 12:00:00 AM
Published date :
Dec 1, 2000, 06:00 AM
Authors :
Zhiqun Tan, Wenli Tu, Hantao Liu, Claude G Wasterlain, Steven S Schreiber, USC Keck Sch of Medicine, Los Angeles, CA; UCLA Sch of Medicine, Los Angeles, CA; Univ of Southern CA Keck Sch of Medicine, Los Angeles, CA.
Rationale: Activation of the p53 DNA damage response pathway has been strongly implicated in seizure-induced neurodegeneration. In non-neural cells p53 abundance is tightly controlled by an inhibitory feedback loop involving the Mdm2 oncoprotein, an E3 ubiquitin ligase that binds to and targets p53 for ubiquitin-mediated degradation. However, the events responsible for p53 stabilization following DNA damage in the central nervous system have not been delineated. In the present study we sought to determine whether the Mdm2-ubiquitin pathway is involved in the mechanism of p53 accumulation and apoptosis in neurons. Methods: Adult male Sprague-Dawley rats were treated with kainic acid (KA, 10 mg/kg, sc); primary neurons were prepared from E18 rat hippocampi and treated with KA (100 M) for 30 min. Immunohistochemistry and Western blotting were performed to evaluate the expression levels of p53, Mdm2 and ubiquitin at specified times following KA treatment in vivo or in vitro. Co-immunoprecipitation of p53-Mdm2 complexes as well as ultrastructural localization of p53 in apoptotic neurons were also performed. Additional studies employed cell-free ubiquitin reconstitution assays and treatment of cultured neurons with ubiquitin antisense oligodeoxynucleotides. Results: Following KA treatment p53 protein accumulated in the nuclei and nucleoli of apoptotic neurons despite the formation of complexes with Mdm2. Notably, p53-Mdm2 complexes were found in neurons exhibiting a striking decrease in ubiquitin expression. p53 degradation was restored to extracts from apoptotic neurons by the addition of exogenous ubiquitin. Damaged neurons also expressed increased amounts of ubiquitin carboxyl-terminal hydrolase which maintains the pool of free ubiquitin. Antisense-mediated downregulation of ubiquitin resulted in an accumulation of both p53 and Mdm2, as well as neuronal apoptosis. Conclusion: Decreased ubiquitin expression is a novel mechanism to abrogate p53 degradation and facilitate p53-mediated apoptosis in the central nervous system.