Rotenone is a naturally derived pesticide that has recently been shown to evoke the behavioral and pathological symptoms of Parkinson's disease in animal models. Though rotenone is known to be an inhibitor of the mitochondrial complex I electron transport chain, little is known about downstream pathways leading to its toxicity. We used human dopaminergic SH-SY5Y cells to study mechanisms of rotenone-induced neuronal cell death. Our results suggest that rotenone, at nanomolar concentrations, induces apoptosis in SH-SY5Y cells that is caspase-dependent. Furthermore, rotenone treatment induces phosphorylation of c-Jun, the c-Jun N-terminal protein kinase (JNK), and the p38 mitogen activated protein (MAP) kinase, indicative of activation of the p38 and JNK pathways. Importantly, expression of dominant interfering constructs of the JNK or p38 pathways attenuated rotenone-induced apoptosis. These data suggest that rotenone induces apoptosis in the dopaminergic SH-SY5Y cells that requires activation of the JNK and p38 MAP kinases and caspases. These studies provide insights concerning the molecular mechanisms of rotenone-induced apoptosis in neuronal cells.

Elucidation of mechanisms regulating cell cycle progression is of fundamental importance for cell and cancer biology. Although several genes and signaling pathways are implicated in G1–S regulation, less is known regarding the mechanisms controlling cell cycle progression through G2 and M phases. We report that extracellular signal–regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinases, is activated at G2–M and required for timely mitotic entry. Stimulation of ERK5 activated nuclear factor κB (NFκB) through ribosomal S6 kinase 2 (RSK2)-mediated phosphorylation and degradation of IκB. Furthermore, selective inhibition of NFκB at G2–M phases substantially delayed mitotic entry and inhibited transcription of G2–M–specific genes, including cyclin B1, cyclin B2, Plk-1, and cdc25B. Moreover, inhibition of NFκB at G2–M diminished mitosis induced by constitutive activation of ERK5, providing a direct link between ERK5, NFκB, and regulation of G2–M progression. We conclude that a novel ERK5–NFκB signaling pathway plays a key role in regulation of the G2–M progression.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type-and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all ␤2 integrins. We designed experiments with the following objectives: to identify which member of the ␤2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced ␤2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.Leukotoxin (Lkt) and lipopolysaccharide produced by Pasteurella (Mannheimia) haemolytica serotype 1 are considered to be the primary virulence factors contributing to lung injury in bovine pneumonic pasteurellosis (BPP) (33,36,38,40), a disease of substantial economic importance to the beef and dairy cattle industries in North America (7,28,39). Lkt is a member of a family of gram-negative bacterial exotoxins termed RTX (for repeats in toxin) cytolysins (3). Although most RTX cytolysins interact with a variety of cell types from many different species (6), cytolysins produced by Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae (ApxIIIA), and P. haemolytica are known to have cell type-and species-specific effects. The leukotoxin (LtxA) of A. actinomycetemcomitans, a human pathogen, interacts only with cells of the lymphocytic and monomyelocytic lineages of humans and some nonhuman primates (23); the Lkt of P. haemolytica, a ruminant pathogen, interacts only with ruminant leukocytes causing activation and cytolysis (4,15,25,33,40). A study by Lally et al. (23) has determined that two RTX cytolysins, ...

Washington 98195-7234 and the §Nencki Institute and the ¶International Institute of Molecular and Cellular Biology, Ksiecia Trojdena 4, Poland Inhibition of glycogen synthase kinase-3␤ (GSK3␤) is one of the mechanisms by which phosphatidylinositol 3-kinase (PI3K) activation protects neurons from apoptosis. Here, we report that inhibition of ERK1/2 increased the basal activity of GSK3␤ in cortical neurons and that both ERK1/2 and PI3K were required for brainderived neurotrophic factor (BDNF) suppression of GSK3␤ activity. Moreover, cortical neuron apoptosis induced by expression of recombinant GSK3␤ was inhibited by coexpression of constitutively active MKK1 or PI3K. Activation of both endogenous ERK1/2 and PI3K signaling pathways was required for BDNF to block apoptosis induced by expression of recombinant GSK3␤. Furthermore, cortical neuron apoptosis induced by LY294002-mediated activation of endogenous GSK3␤ was blocked by expression of constitutively active MKK1 or by BDNF via stimulation of the endogenous ERK1/2 pathway. Although both PI3K and ERK1/2 inhibited GSK3␤ activity, neither had an effect on GSK3␤ phosphorylation at Tyr-216. Interestingly, PI3K (but not ERK1/2) induced the inhibitory phosphorylation of GSK3␤ at Ser-9. Significantly, coexpression of constitutively active MKK1 (but not PI3K) still suppressed neuronal apoptosis induced by expression of the GSK3␤(S9A) mutant. These data suggest that activation of the ERK1/2 signaling pathway protects neurons from GSK3␤-induced apoptosis and that inhibition of GSK3␤ may be a common target by which ERK1/2 and PI3K protect neurons from apoptosis. Furthermore, ERK1/2 inhibits GSK3␤ activity via a novel mechanism that is independent of Ser-9 phosphorylation and likely does not involve Tyr-216 phosphorylation.

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Administration of rotenone to rats reproduces many features of Parkinson's disease, including dopaminergic neuron degeneration, and provides a useful model to study the pathogenesis of Parkinson's disease. However, the cell death mechanisms induced by rotenone and potential neuroprotective mechanisms against rotenone are not well defined. Here we report that rotenone-induced apoptosis in human dopaminergic SH-SY5Y cells is attenuated by pretreatment with several growth factors, most notably basic fibroblast growth factor (bFGF). bFGF activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase (PI3-kinase) pathways in SH-SY5Y cells. Ectopic activation of ERK1/2 or PI3-kinase protected against rotenone, whereas inhibition of either pathway attenuated bFGF protection. Reducing the expression of the proapoptotic protein Bcl-2-associated death protein (BAD) by small interfering RNA rendered SH-SY5Y cells resistant to rotenone, implicating BAD in rotenone-induced cell death. Interestingly, bFGF induced a long-lasting phosphorylation of BAD at serine 112, suggesting BAD inactivation through the ERK1/2 signaling pathway. Moreover, primary cultured dopaminergic neurons from mesencephalon were more sensitive to rotenone-induced cell death than nondopaminergic neurons in the same culture. The loss of dopaminergic neurons was blocked by bFGF, an inhibition dependent on ERK1/2 and PI3-kinase signaling. These data suggest that rotenone-induced dopaminergic cell death requires BAD and identify bFGF and its activation of ERK1/2 and PI3-kinase signaling pathways as novel intervention strategies to block cell death in the rotenone model of Parkinson's disease.

Isatis indigotica posses a valuable virucidal effect in disease control of pseudorabies virus infection in swine.