Membrane depolarization-mediated survival of sympathetic neurons occurs through both phosphatidylinositol 3-kinase- and CaM kinase II-dependent pathways

doi:10.1016/S0006-8993(00)02284-8

Brain Research

Volume 866, Issues 1–2, 2 June 2000, Pages 218-226

https://doi.org/10.1016/S0006-8993(00)02284-8 Get rights and content

Abstract

It has been well established that the NGF-mediated survival of sympathetic neurons in culture occurs through the phosphatidylinositol (PI) 3-kinase/Akt-dependent pathway. In contrast, the mechanism by which membrane depolarization promotes neuronal survival independently of NGF remains unresolved. Here we show that LY294002, a specific inhibitor of PI 3-kinase, induced cell death of sympathetic neurons under depolarizing conditions with elevated K⁺ (IC₅₀=∼30 μM). Interestingly, lower concentrations of this agent (≤10 μM) were sufficient to suppress Akt phosphorylation at Ser-473, a putative downstream target of PI 3-kinase, under these conditions. We also show that KN-62, a specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) suppressed depolarization-mediated survival in a does-dependent manner (IC₅₀=∼2 μM) that paralleled attenuation of sustained levels of intracellular Ca²⁺ evoked by depolarization. This IC₅₀ value is greater than that for CaMKII (∼0.8 μM). These findings led us to hypothesize that depolarization-mediated survival occurs through both the PI 3-kinase/Akt and the CaMKII pathways. Indeed, combined treatment with LY294002 (25 μM) and KN-62 (0.5 μM) dramatically abolished depolarization-mediated survival, whereas each alone did not significantly attenuate it. Under these conditions, KN-62 neither impaired sustained levels of intracellular Ca²⁺, nor inhibited the phosphorylation of Akt. It is thus likely that PI 3-kinase and CaMKII independently promote the membrane depolarization-mediated survival of sympathetic neurons in culture.

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.

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Research reportMembrane depolarization-mediated survival of sympathetic neurons occurs through both phosphatidylinositol 3-kinase- and CaM kinase II-dependent pathways

Abstract

Introduction

Section snippets

Cell culture

LY294002 suppresses Akt phosphorylation and eventually neuronal survival of sympathetic neurons under depolarizing conditions

Discussion

Acknowledgements

Cell

FEBS Lett.

Biochem. Biophys. Res. Commun.

Neuroscience

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Dev. Brain Res.

Neuropharmacology

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

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

Research report
Membrane depolarization-mediated survival of sympathetic neurons occurs through both phosphatidylinositol 3-kinase- and CaM kinase II-dependent pathways