Compromised mitochondrial function leads to increased cytosolic calcium and to activation of MAP kinases

Abstract

We have investigated in rat pheochromacytoma PC12 cells the activation of the mitogen-activated protein kinases ERK1 and ERK2 by the mitochondrial uncoupler carbonyl cyanide p-(trif luoromethoxy)phenylhydrazone (FCCP). This treatment slowly decreases ATP levels to 30% of control, whereas the internal calcium level rises very rapidly to 250% of control, derived from internal stores. Tyrosine phosphorylation of ERK1 and ERK2 increases gradually, starting after 5 min of treatment, to reach a maximum at 30 min; the kinase activity reaches 250% when measured after 1 hr of treatment. The drop in ATP levels is slower still. The neurodegenerative chain of events in Alzheimer disease (AD) are unknown. Recent evidence indicates that mitochondrial dysfunction, defects in energy metabolism, and excitotoxicity might be involved (1, 2). For example, both mitochondrial DNA deletions (3, 4) and defects in mitochondrial oxidative phosphorylation (5, 6) have been observed in the aged and the AD brain. Mitochondria are the site of ATP production and also play a role in intracellular calcium homeostasis because they can sequester large quantities of calcium (7). Effective buffering of cytosolic calcium is critical to neuronal survival because elevated cytosolic calcium would stimulate glutamate release, resulting in activation of Nmethyl-D-aspartate receptors, which in turn would result in massive Ca 2ϩ influx and cell damage and death. Several lines of evidence indicate that calcium homeostasis is altered in aging and AD (8-10). A reduction of Ca 2ϩ storage in mitochondria has been detected in old rats (11). Calcium release from the inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum (ER) pool (12) and from an additional ionophoresensitive pool (13) is enhanced in fibroblasts from AD patients. These results suggest that an age-associated increase in internal calcium concentration may be relevant to such neurodegenerative disorders. In fact, neurofibrillary tangle-like antigenic changes and ␤-amyloidosis can be elicited by glucose deprivation and calcium influx in cultured hippocampal neurons (14), skin fibroblasts (15), human neuroblastoma cells (16), and COS cells (17).The molecular mechanism that links mitochondrial dysfunction to neuronal death is not clear. Previously, we demonstrated (18) that treatment of PC12 cells with the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) led to hyperphosphor ylation of neurofilament (NF) and Tau in these cells, suggesting an activation of NF and͞or Tau kinases. We undertook this study to investigate the mechanism involved. We examined the role of increased cytosolic calcium in the activation of ERK1 and ERK2 in PC12 cells. Our results indicate that FCCP treatment of PC12 cells leads to calcium release from mitochondrial stores, and also to phosphorylation͞activation of the mitogenactivated protein (MAP) kinases ERK1 and ERK2. The possible implications of these effects in AD are discussed.

Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

“…The activation of MAP kinase in A549, but not in C2C12 cells, probably reflects cell-specific differences in mitochondrial stress-induced activation of different signal pathways. Our results are consistent with a previous study (Luo et al, 1997), which showed that ERK1 and ERK2 were induced in phechromocytoma PC12 cells treated with FCCP, a mitochondria-specific ionophore, which also caused an increase in steady state [Ca 2+ ]. Additionally, increased mitochondrial H 2 O 2 , possibly representing mitochondrial stress, also induced MAP kinases in Hela cells (Nemoto et al, 2000).…”

Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission

“…Uncoupling by FCCP reduced ATP by 30 -35% (Fig. 5B), which is in agreement with previous report (42). Subsequent increases in O 2 consumption by e K ϩ had no effect on the ATP level.…”

Mitochondrial Abnormalities in Alzheimer's Disease

“…Other changes of AD could very well be linked to mitochondria because blockage of mitochondrial energy production shifts amyloid ␤-protein precursor metabolism to the production of more amyloidgenic forms of amyloid-␤ (Gabuzda et al, 1994), induces the production of A68 antigen (Blass et al, 1990), and activates the mitogen-activated protein kinase pathway (Luo et al, 1997;Perry et al, 1999;Zhu et al, 2000Zhu et al, , 2001, all features of AD.…”