Calcineurin Activity Is Regulated Both by Redox Compounds and by Mutant Familial Amyotrophic Lateral Sclerosis‐Superoxide Dismutase

Abstract

Calcineurin (CN) is a protein phosphatase involved in a wide range of cellular responses to calciummobilizing signals, and a role for this enzyme in neuropathology has been postulated. We have investigated the possibility that redox modulation of CN activity is relevant to neuropathological conditions where an imbalance in reactive oxygen species has been described. We have monitored CN activity in cultured human neuroblastoma SH-SY5Y cells and obtained evidence that CN activity is promoted by treatment with ascorbate or dithiothreitol and impaired by oxidative stress. Evidence for the existence of a redox regulation of this enzyme has been also obtained by overexpression of wild-type antioxidant Cu,Zn superoxide dismutase (SOD1) that promotes CN activity and protects it from oxidative inactivation. On the contrary, overexpression of mutant SOD1s associated with familial amyotrophic lateral sclerosis (FALS) impairs CN activity both in transfected human neuroblastoma cell lines and in the motor cortex of brain from FALS-transgenic mice. These data suggest that CN might be a target in the pathogenesis of SOD1-linked FALS. Key Words: Calcineurin-Familial amyotrophic lateral sclerosis-Superoxide dismutase-Neurodegeneration-Redox regulation-Reactive oxygen species. J. Neurochem. 75, 606 -613 (2000).Calcineurin (CN), also known as protein phosphatase 2B, is a heterodimeric enzyme composed of a catalytic A-subunit (molecular mass ϳ60 kDa) and a myristoylated regulatory B-subunit (molecular mass ϳ19 kDa) (Klee et al., 1988). The A-subunit has four distinct domains: the catalytic domain, the B-subunit binding domain, the Ca 2ϩ /calmodulin complex binding domain, and the auto-inhibitory domain. The B-subunit is structurally related to calmodulin, containing four EF-hand Ca 2ϩ binding loops. The A-subunit binds one zinc and one iron atom at the active site, which probably serve to position water molecules by acting as nucleophiles in the attack on the phosphoester bond (Griffith et al., 1995;Kissinger et al., 1995). The auto-inhibitory domain masks the metal ions at the active site; the activation of CN by Ca 2ϩ /calmodulin probably results from the displacement of this domain, resulting in exposure of the catalytic center. In this "open" conformation, CN is sensitive to inactivation by small (undefined) molecules (Wang et al., 1996). CN is enriched in neural tissue and comprises Ͼ1% of the total protein in the nervous system (Klee et al., 1988), where it acts as a Ca 2ϩ -and calmodulin-dependent serine/threonine protein phosphatase involved in a wide range of cellular responses to calcium-mobilizing signals (Yakel, 1997). Its role in regulating neuronal excitability by controlling the activity of ion channels, the release of neurotransmitter and hormone, synaptic plasticity, and gene transcription is well documented (Yakel, 1997). A role for CN in cell cycle regulation (Davis et al., 1997) and in Ca-dependent apoptosis, mediated by interaction of CN with Bcl-2 (Reed, 1997) and by dephosphorylation of BAD (Wang...

Axonal transport of neurofilaments in normal and disease states

“…Recently, deregulated cdk5/p35 has been described in mutant SOD1 mice [114] and we have shown that glutamate, a proposed pathogenic mechanism for several neurodegenerative diseases, activates members of the MAPK/SAPK family and causes a slowing of neurofilament transport and increased cell body phosphorylation of neurofilaments [47]. Other studies have shown that SOD1 influences the activity of calcineurin, a neurofilament phosphatase [115,116]. Thus, there is increasing evidence to link known pathogenic processes involved in neurodegenerative diseases with signal transduction cascades that regulate neurofilament phosphorylation.…”

Synaptic sprouting increases the uptake capacities of motoneurons in amyotrophic lateral sclerosis mice

“…However, the increase in intracellular Ca 2ϩ levels in the G93A mice (31)(32)(33) argues against this hypothesis because the interaction between calcineurin and dynamin is optimal for a small range of intracellular Ca 2ϩ concentration (30). Moreover, calcineurin activity was reported to be either decreased or unchanged in cells overexpressing SOD1 mutants (34,35). Alternatively, the higher level of endocytosis in G93A mice could be due to the activation of GAP-43, a growth-associated protein present in growth cones and synaptic terminals, which has been implicated in nerve sprouting and endocytosis (36,37).…”

“…During activity-dependent increases in Ca 2ϩ levels, GAP-43 interacts with rabaptin-5, a protein involved in endocytosis (37). Because GAP-43 is negatively regulated by the phosphatase activity of calcineurin (38), the phosphorylation state of GAP-43 may increase secondarily to possible calcineurin inhibition in G93A mice reported in cells overexpressing SOD1 mutants (34). This hypothesis is supported further by the protective effect of wt SOD on Ca 2ϩ ͞calmodulin-dependent calcineurin inactivation (39).…”

Oxidative Stress Induces Neuronal Death by Recruiting a Protease and Phosphatase-gated Mechanism

“…This phosphatase has been previously described to regulate dephosphorylation of pCREB in hippocampal neurons (43), and calcineurin has been implicated in neuronal apoptosis (19,20) and in neurodegenerative diseases (18,60). However, activation of this phosphatase during oxidative stress is not in line with other reports that superoxide-induced oxidative stress results in an inhibition of calcineurin activity (61)(62)(63) can impinge on different signaling pathways (51); in addition, cell and tissue type may be important in determining these differences.…”