Signal-transduction pathways in the CNS provide key mechanisms for cellular responses to environmental stresses. Oxidant-stresses are operative in the aging brain with infarction and Alzheimer's disease. MAP kinases mediate these inputs to downstream targets, such as transcription factors. The result is either regeneration and protection, or cell death. DENN-MADD, a GDP/GTP exchange factor expressed in neurons has basal functions mediating release of neurotransmitters from presynaptic terminals. DENN-MADD, identified by two-hybrid screening of a human brain cDNA library using JNK3 as bait, is expressed in neurons as a 200KD protein. Functional domains include a nuclear localization signal,JNK binding domain, leucine-zipper and a death domain. Under oxidant stress conditions, DENN-MADD is translocated from the cytoplasm to the nucleolus, and the cell ultimately dies. To compare basal and stress-induced effects, we will first examine, interactions of JNK and other MAPKs with DENN-MADD, including binding and phosphorylation. Using cDNA deletion constructs transfected into neuronal cultures, mechanisms of nuclear translocation, including masking of the putative nuclear localization signal and interactions with other nucleolar-associated proteins will be examined. Second, the DENN-MADD death domain is known to bind to a death domain of the TNF receptor alpha-1, part of an apoptosis pathway. Interactions of DENN-MADD with TNFR and other death domain proteins will be compared under stress (hypoxia, A-beta). Third, effects of nuclear translocation of DENN-MADD on basal neuronal functions, specifically synaptic vesicle release will be tested to determine if stress results in reduced acetylcholine release. Fourth, possible synergistic stress effects of A-beta and hypoxia on DENN-MADD mediated cell death will be compared immunocytochemically in primary CNS tissue cultures and directly in brain tissues from AD, CNS infarction and normal, aged patients. Neurons vulnerable or resistant to oxidant stress will be compared for DENN-MADD nuclear translocation. This pivotal role of DENN-MADD provides a model for selective neuronal vulnerability in response to oxidant stress.
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