Guanosine 3',5'-cyclic monophosphate mediated inhibition of cell death induced by nerve growth factor withdrawal and beta-amyloid: protective effects of propentofylline.

Apoptotic cell death has been implicated in Alzheimer's disease pathology and amyloid peptide induced neurotoxicity. We investigated the survival promoting effects of Propentofylline in two models of apoptotic cell death, nerve growth factor withdrawal and beta-amyloid mediated cell death in nerve growth factor differentiated rat pheochromocytoma cell lines. The increase in cell death as measured by lactate dehydrogenase release in response to nerve growth factor withdrawal was suppressed by nitric oxide donor S-nitroso-N-acetylpenicillamine (12.5 to 200 microM) and by 8-bromoguanosine-3',5'-cyclic monophosphate (1.25 to 10mM). Both agents decreased cell death mediated by 25 microM beta-amyloid, suggesting that the protective mechanism involves guanosine -3', 5'-cyclic monophosphate. In support of this hypothesis we can show that S-nitroso-N-acetylpenicillamine increases intracellular levels of guanosine -3',5'-cyclic monophosphate in pheochromocytoma cell lines 3 to 8 fold.Propentofylline, a phosphodiesterase inhibitor, has previously demonstrated neuroprotective activity in stroke models and is a potential candidate for therapeutic treatment in neurodegenerative diseases. The present findings support this claim by providing evidence that Propentofylline has protective effects in both nerve growth factor withdrawal and beta-amyloid mediated cell death. Lactate dehydrogenase release was significantly reduced and caspase-3-like activity was attenuated after cotreatment with Propentofylline. Furthermore Propentofylline dose responsively increases intracellular guanosine-3',5'-cyclic monophosphate levels over the same dose range that provided protection. We hypothesized that guanosine-3',5'-cyclic monophosphate is a key mediator of neuroprotection under these conditions.

Involvement of cell cycle elements, cyclin-dependent kinases, pRb, and E2F x DP, in B-amyloid-induced neuronal death.

Previous evidence by others has indicated that a variety of cell cycle-related molecules are up-regulated in brains of Alzheimer's disease patients. The significance of this increase, however, is unclear. Accordingly, we examined the obligate nature of cyclin-dependent kinases and select downstream targets of these kinases in death of neurons evoked by B-amyloid (AB) protein. We present pharmacological and molecular biological evidence that cyclin-dependent kinases, in particular Cdk4/6, are required for such neuronal death. In addition, we demonstrate that the substrate of Cdk4/6, pRb/p107, is phosphorylated during AB treatment and that one target of pRb/p107, the E2F x DP complex, is required for AB-evoked neuronal death. These results provide evidence that cell cycle elements play a required role in death of neurons evoked by AB and suggest that these elements play an integral role in Alzheimer's disease-related neuronal death.

Amyloid beta-peptides inhibit Na+/K+-ATPase: tissue slices versus primary cultures.

Abeta1-40 (20 microM) has been reported to selectively inhibit Na+/K+-ATPase activity in rat primary hippocampal cultures after 2-6 days of exposure. We expanded these studies to include Abeta's effects on Na+/K+-ATPase activity in rat primary cortical cultures and hippocampal slices, and we correlated these effects with estimates of cell survival in rat brain primary cultures. Using optimized assay conditions, a 5-day exposure to 50 microM Abeta 25-35, 20 microM Abeta 1-40, and 20 microM Abeta 1-42 decreased Na+/K+-ATPase activity in rat primary cortical cultures 66%, 60%, and 22%, respectively. Abeta 25-35 (50 microM) at 24 h was the only condition that caused inhibition of Na+/K+-ATPase activity in the absence of cell death, defined as an extracellular shift in the localization of the cytoplasmic enzyme lactate dehydrogenase (LDH). We also found that hippocampal slices were sensitive to Abeta, exhibiting a 40-60% reduction in membrane Na+/K+-ATPase activity when exposed to 1-30 nM of Abeta 1-40 for 60 min. This inhibition was not readily reversible, as it withstood homogenization and repeated dilution and centrifugation. Additionally, this inhibition occurred only after amyloid incubation with intact hippocampal slices, not with disrupted membranes. The inhibition of Na+/K+-ATPase in brain slices by physiological, low nM concentrations of Abeta 1-40 is consistent with effects on neurotransmitter release and intrasynaptosomal calcium responses.