Cyclosporin A regulates the levels of cyclophilin A in neuroblastoma cells in culture.

Cyclophilin A (CyP-A), a member of a highly conserved family of proteins, immunophilins, is the major intracellular receptor for the immunosuppressive drug, cyclosporin A (CsA). CyP-A is widely expressed in many tissues, but is found in the highest concentration in brain tissues and may perform critical neuronal functions. CsA is a known neurotoxin. Therefore, understanding the regulation of CyP-A levels in nerve cells, particularly by CsA, is important. We have utilized murine neuroblastoma (NB) cells as an experimental model to investigate this issue. Our results show that CsA alone was sufficient to induce morphological differentiation in undifferentiated NB cells and to increase CyP-A levels as determined by immunostaining. However, inducing terminal differentiation by elevating adenosine 3',5'-cyclic monophosphate (cAMP) levels using either 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724), an inhibitor of cyclic nucleotide phosphodiesterase, or prostaglandin E1 (PGE1), a stimulator of adenylate cyclase, was not sufficient to increase CyP-A levels. CsA was required to increase CyP-A levels in both RO20-1724- and PGE1-induced differentiated NB cells. Increases in CyP-A levels, however, occurred without any change in the expression of the CyP-A gene as determined by reverse-transcriptase polymerase-chain reaction analysis using (CyP-A)-specific primers. These results suggest that CsA regulates the level of its own binding protein, CyP-A, in both undifferentiated and cAMP-induced differentiated NB cells in culture.

Prostaglandins as putative neurotoxins in Alzheimer's disease.

Chronic inflammatory reactions in the brain appear to be one of the primary etiological factors in the pathogenesis of Alzheimer's disease (AD). This is supported by the fact that the secretory products of inflammatory reactions, which include cytokines, complement proteins, adhesion molecules, and free radicals, are neurotoxic. We have recently reported that prostaglandins (PGs), which are also released during inflammatory reactions, cause rapid degenerative changes in differentiated murine neuroblastoma cells (NB) in culture. PGA1 is more effective than PGE1. Similar observations were made in a primary culture of fetal rat hippocampal cells. Epidemiological and clinical studies on AD also support the involvement of PGs in neuronal degeneration. Thus, we propose a hypothesis that PGs are one of the major extracellular signals that initiate neuronal degeneration, which is mediated by intracellular signals such as the beta-amyloid peptide (Abeta) and ubiquitin, since the levels of these proteins are increased by PG treatment. We further suggest that adenosine 3', 5'-cyclic monophosphate (cAMP) is one of the factors that regulate the levels of both Abeta and ubiquitin in NB cells. Increases in the level of Abeta in NB cells following an elevation of intracellular cAMP levels appear to be due to an increase in the rate of processing of the amyloid precursor protein (APP) rather than an increase in the expression of APP. The mechanisms underlying Abeta-induced neuronal degeneration have been under intense investigation, and several mechanisms of action have been proposed. We postulate that PG-induced elevation of Abeta may lead to an increased binding of Abeta to the 20S proteasome, resulting in a reduction of 20S proteasome-mediated degradation of ubiquitin-conjugated proteins. This is predicted to lead to an increase in an accumulation of abnormal proteins, which ultimately contribute to neuronal degeneration and death. Based on our hypothesis and on studies published by others, we propose that a combination of nonsteroidal anti-inflammatory drugs, which inhibit the synthesis of PGs, and antioxidant vitamins, which quench free radicals and both of which have been recently reported to be of some value in AD treatment when used-individually, may be much more effective in the prevention and treatment of AD than the individual agents alone.

Overexpression of the protein kinase Pak1 suppresses yeast DNA polymerase mutations.

This article presents the identification and characterization of the PAK1 gene of Saccharomyces cerevisiae, and the biochemical characterization of the protein kinase activity that it encodes. Overexpression of the PAK1 gene product suppresses temperature-sensitive mutations of the poll (cdc 17) gene, which encodes DNA polymerase alpha. Overexpression and suppression can be achieved either by expressing PAK1 from a high-copy-number plasmid, or by GAL1-induced transcription of PAK1. Gene disruption of PAK1 indicates that it is not an essential gene. The PAK1 gene encodes a protein with a kinase consensus domain. By deletion analysis and site-directed mutagenesis, we demonstrate that the complete and active kinase consensus domain is required for suppression. A glutathione-S-transferase (GST)-Pak1 fusion protein, overproduced in bacteria, can be purified in an active form with glutathione affinity beads or by immunoprecipitation. Thus, other protein subunits of Pak1 are not required for its activity. In vitro protein kinase assays show that GST-Pak1 can autophosphorylate, and can phosphorylate casein as an exogenous substrate. The phenotype of the suppressed cdc17-1 cells indicates that Pak1 suppression is inefficient and does not restore the wild-type phenotype. Pak1 suppression requires Rad9 function, but Pak1 does not affect Rad9 function. Overexpression of PAK1 does not enhance the expression of the POL1 gene. Pak1 may function by modifying and partially stabilizing thermolabile DNA polymerases, perhaps during DNA repair, because pak1 mutant cells are caffeine sensitive.