Research reportMembrane depolarization-mediated survival of sympathetic neurons occurs through both phosphatidylinositol 3-kinase- and CaM kinase II-dependent pathways
Introduction
Target-derived neurotrophic factors play a pivotal role in promoting the survival of many types of mature neurons. The mechanism by which these factors promote neuronal survival has been under intense investigation. Using wortmannin as a phosphatidylinositol (PI) 3-kinase inhibitor, Yao and Cooper [34] have demonstrated that PI 3-kinase is involved in the NGF-mediated survival of pheochromocytoma PC12 cells. Since then, direct evidence has been accumulated by transfecting neurons with dominant negative forms of PI 3-kinase, providing supporting evidence that PI 3-kinase plays a major role in the neurotrophic factor-mediated survival of many types of neurons including sympathetic neurons [4]. Moreover, Akt (also called RAC kinase or protein kinase B), a putative downstream target of PI-3 kinase, has also been reported to function as an essential mediator in trophic factor-dependent neuronal survival.
The electrical activity of neurons is also known to regulate neuronal survival through trophic factor-dependent or -independent pathways. For example, elevated potassium causes membrane depolarization which induces a sustained increase in intracellular calcium concentration ([Ca2+]i) resulting from the opening of voltage-gated calcium channels [12], [19]. Although the sustained increase in [Ca2+]i levels is important for neuronal survival, the mechanisms by which the calcium increase promotes neuronal survival remains elusive, whereas those of trophic factor-mediated survival are well established. In cerebellar granule neurons, PI 3-kinase but not extracellular signal-regulated kinase (ERK) has been reported to be an important mediator of neuronal survival promoted by membrane depolarization [22]. By contrast, other studies have not provided evidence supporting the involvement of PI 3-kinase in the depolarization-mediated survival of cerebellar granule neurons [7], [8]. Recently, studies with dominant negative PI 3-kinase and Akt show that the PI 3-kinase/Akt pathway is required for the survival of sympathetic neurons under depolarizing conditions [5].
In contrast, there is a consensus that elevated levels of [Ca2+]i under depolarizing conditions activate calmodulin, leading to the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Indeed, CaMKII is important for the survival of cerebellar granule neurons via membrane depolarization [14]. Moreover, the study using spinal cord motoneurons has revealed that calmodulin, but neither PI 3-kinase nor ERK, is essential for the depolarization-promoted survival [26]. It has been also reported that the depolarization-mediated survival of sympathetic neurons is highly dependent on CaMKII [30].
In this report, we have addressed the question as to whether PI 3-kinse/Akt and CaMKII act independently or sequentially to promote the membrane depolarization-mediated survival of sympathetic neurons in culture. Here, we show that blockade of both the PI 3-kinase/Akt and CaMKII pathways completely abolishes the survival promoted by depolarization, whereas inhibition of either pathway alone does not impair the survival-promoting effect of depolarization.
Section snippets
Cell culture
Cultured sympathetic neurons were prepared from rat superior cervical ganglia (SCG) dissected from neonatal rats (Sprague–Dawley) as previously described [19]. Briefly, dissected ganglia were treated with 1 mg/ml of collagenase (Worthington Biochemical Corp., Lakewood, NJ, USA) at 37°C for 25 min. After washing, these ganglia were triturated and suspended in Eagle’s minimum essential medium (MEM; Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS; Trace
LY294002 suppresses Akt phosphorylation and eventually neuronal survival of sympathetic neurons under depolarizing conditions
It has been reported that LY294002 specifically suppresses PI 3-kinase, a putative downstream factor of TrkA, the NGF receptor [31], and eventually inhibits NGF-dependent survival of sympathetic neurons [4]. We tested the effect of LY294002 on the depolarization-mediated survival of SCG neurons and found that the survival-promoting effect of elevated K+ (45 mM) was effectively abolished in a dose-dependent manner (IC50=∼30 μM; Fig. 1A). This result indicates that PI 3-kinase promotes the
Discussion
Many types of neurons from both the central and peripheral nervous systems depend on trophic factors for their survival, such as NGF, brain-derived neurotrophic factor, and insulin-like growth factor 1. Several recent reports have provided evidence that trophic factors promote neuronal survival through the PI 3-kinase dependent pathway [2], [7], [8], [22], [24]. However, less is known concerning the survival-promoting mechanisms mediated by membrane depolarization. In this report, we provided
Acknowledgements
This work is partly supported by the Grant-in-Aid No. 96-14 of the Program for the Promotion of Fundamental Studies in Health Sciences of Japan.
References (35)
- et al.
Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery
Cell
(1997) - et al.
High affinity calmodulin target sequence in the signaling molecule PI 3-kinase
FEBS Lett.
(1998) - et al.
Akt phosphorylation site found in human caspase-9 is absent in mouse caspase-9
Biochem. Biophys. Res. Commun.
(1999) - et al.
Promotion of granule cell survival by high K+ or excitatory amino acid treatment and Ca2+/calmodulin-dependent protein kinase activity
Neuroscience
(1993) - et al.
Cadmodulin activates phosphatidylinositol 3-kinase
J. Biol. Chem.
(1997) - et al.
Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase
J. Biol. Chem.
(1994) - et al.
Inhibition of phosphatidylinositol 3-kinase activity blocks depolarization- and insulin-like growth factor I-mediated survival of cerebellar granule cells
J. Biol. Chem.
(1997) - et al.
Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway
J. Biol. Chem.
(1998) - et al.
Involvement of phosphatidylinositol-3 kinase in prevention of low K+-induced apoptosis of cerebellar granule neurons
Dev. Brain Res.
(1997) - et al.
Ca/calmodulin-dependent kinase II inhibitor KN62 attenuates glutamate release by inhibiting voltage-dependent Ca2+-channels
Neuropharmacology
(1995)
KN-62, 1-[N,O-Bis(5-isoquinokinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II
J. Biol. Chem.
Measurement of cytosolic free Ca2+ with quin2
Methods Enzymol.
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002)
J. Biol. Chem.
Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L)
Cell
The p75 neurotrophin receptor mediates neuronal apoptosis and is essential for naturally occurring sympathetic neuron death
J. Cell Biol.
Regulation of cell death protease caspase-9 by phosphorylation
Science
Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B
Nature
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