REGULATION OF APOPTOSIS BY THE SEROTONIN 1A RECEPTOR

点击翻译按钮获取中文摘要

Aging is concomitant with progressive loss of neurons mainly through the slow, suicidal process of apoptosis. While the resulting loss of physical and cognitive functions have been studies by many groups, only sparse information is available on receptor-mediated, neuroprotective signaling cascades which undergo a significant decline during aging. Earlier studies have shown that the serotonin 1A receptor (5-HT1A-R) undergoes an age-related decline, however, the possibility that this loss of 5-HT1A-R expression could be a reason for neuronal apoptosis has not been addressed. Preliminary data show that the agonist-bound 5- HT1A-R causes inhibition of apoptosis by stimulating the mitogen activated kinase (MAPK) via intermediate proteins Ras and Raf-1. However apoptosis has been directly linked to increased Cytochrome c release from mitochondria, proteolytic activation of the cytosolic, interleukin 1beta-convertase (ICE)-like protease CPP32 and subsequent breakdown of cytoskeletal proteins (e.g. Fodrin) and nuclear DNA. The relationship between receptor mediated activation of MAPK and the pathways leading to CPP32 processing and activation is not known. This pilot project is designed to yield information which will form the foundation for a broader study to link these two events. Preliminary data showing 5-HT1A-R mediated inhibition of apoptosis, CPP32 processing and Fodrin cleavage in neuronal cells suggest that agonist binding to the 5-HT1A-R may cause an activation of the mitochondrial protein Bcl-2 which inhibits Cytochrome c release from mitochondria. This Bcl-2 activation could be through phosphorylation mediated inactivation of the death agonist protein, Bad, which before phosphorylation, binds to and inactivates Bcl-2. Serine-phosphorylation of Bad could be the result of 5-HT1A-R mediated activation of the serine/threonine kinase MAPK. Alternatively, the Bcl-2 binding protein Raf-1, upon MAPK mediated serine-phosphorylation and detachment from the plasma membrane, could bind to and transport the phosphoserine-binding, cytosolic protein 14-3-3 to the Bcl-2: Bad complex located on the mitochondria. Subsequent to its binding to Bcl-2, Raf-1 could cause serine- phosphorylation of Bad and prepare it for association with 14-3-3 and removal from Bcl-2, thus restoring Bcl-2 activity. For either model, 5-HT1A-R mediated increase in phosphorylation of Bad and association of phosphorylated Bad with 14-3-3 will be tested by 32PO43-labeling, immunoprecipitation and immunoblot analysis. MAPK mediated, direct phosphorylation of Bad will be tested through a time course analysis, since 5-HT1A-R mediated activation of MAPK occurs rapidly within 3 min. An indirect mechanism via phosphorylation of Raf-1, binding of phospho- Raf-1 to the Bcl-2:Bad complex and increase in phosphorylation of Bad should be a slower process requiring several protein-protein associations. Increased Raf-1 phosphorylation and Raf-1:Bcl-2 binding upon agonist activation of the 5-HT1A-R will be tested by 32PO43- labeling, coimmunoprecipitation of Raf-1 with Bcl-2 using Bcl-2 antibody and immunoblotting. The model confirmed will form the basis for future studies to elucidate the complete pathway from 5-HT1A-R activation to neuroprotection.