Elsevier

Brain Research

Volume 929, Issue 1, 1 March 2002, Pages 87-95
Brain Research

Research report
Protein kinase C activation is required for the lead-induced inhibition of proliferation and differentiation of cultured oligodendroglial progenitor cells

https://doi.org/10.1016/S0006-8993(01)03385-6Get rights and content

Abstract

Lead (Pb) is a common neurotoxicant of major public health concern. Previous studies revealed that cultured oligodendrocyte progenitor cells (OPCs) are highly vulnerable to Pb toxicity. The present study examines the effect of Pb on the survival, proliferation and differentiation of OPCs in vitro. Dose–response studies showed that ≥5–10 μM Pb is cytotoxic to OPCs within 24 h. However, 1 μM of Pb was found to inhibit the proliferation and differentiation of OPCs without affecting cell viability. Pb markedly decreased the proliferative capability of OPCs and inhibited cell-intrinsic lineage progression of OPCs at a late progenitor stage. The Pb-induced decrease of proliferation and differentiation was abolished by inhibition of protein kinase C (PKC) with bisindolylmaleimide I, while the effect of the PKC-activating agent phorbol-12,13-didecanoate was potentiated by Pb. Furthermore, Pb exposure of OPCs caused the translocation of PKC from the cytoplasm to membrane without an increase in total cellular PKC enzymic activity. These results indicate that Pb inhibits the proliferation and differentiation of oligodendrocyte lineage cells in vitro through a mechanism requiring PKC activation.

Introduction

Oligodendrocytes are the myelin-forming cells in the central nervous system (CNS). The oligodendrocyte progenitor cells (OPCs) are well characterized both in vivo and in vitro, and found in both the developing and adult brains [7], [19], [20], [26], [36], [37], [41]. OPCs are a unique population of glial cells that appear to play a critical role in brain development. While OPCs are capable of differentiating into myelin-competent oligodendrocytes, they also participate in neuronal development and function, synaptic activity and neurotransmission. Evidence from in vitro studies has demonstrated that OPCs can influence synaptic efficacy [35], support neuronal survival [42], enhance axonal growth [40], and produce factors that induce neuronal sodium channel clustering [22]. In vivo studies have shown that OPCs form functional glutaminergic synapses with hippocampal CA1 neurons [10]. These properties of OPCs, which can influence brain development, may well affect cognitive performance. OPCs, originally termed O-2A progenitors because of the ability to develop into either oligodendrocytes or type-2 astrocytes depending upon the culture conditions [37], appear to be reprogramable to neural stem cells capable of giving rise to neurons, oligodendrocytes or type-1 astrocytes [24]. They can proliferate to supply new progenitors, differentiate to provide new oligodendrocytes for replacement of those lost, or undergoing apoptosis in response to CNS injury [26].

Relatively low-level environmental exposure to lead (Pb) by children is a major public health concern and has been associated with cognitive and behavioral deficits in affected individuals [9], [15], [21]. Both neurons [3], [13], [14], [46] and astrocytes [25], [44], [45] are cellular targets for the metal. Neurons have been long thought to be the primary cellular target for Pb toxicity in the brain and to be responsible for the Pb-induced cognitive and neurobehavioral deficits, while astroglia may function as a ‘lead sink’ to protect neurons from damage caused by Pb [44], [45]. The role of oligodendrocyte lineage cells in this context has not been appreciated. Recently, we demonstrated that in vitro cultured oligodendrocyte lineage cells are susceptible to the toxicity of environmentally relevant concentrations of Pb [16], [18]. Given the potential role of OPCs in neuronal development and function [10], [22], [35], [40], [42], in addition to myelin synthesis and maintenance, it is possible that the effect of Pb on OPC function, survival and developmental potential may be a contributing mechanism resulting in cognitive and neurobehavioral deficits in exposed individuals. The present study investigates the involvement of protein kinase C (PKC) in Pb-mediated effects on OPC proliferation and differentiation. These aspects of OPC biology are under the control of a number of growth factors, such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) [19], [20], [33], [34], [36]. PDGF induces early progenitor cells to proliferate for a defined number of divisions, thereby preventing premature differentiation. Basic FGF is a mitogen for late progenitors and arrests terminal differentiation at that stage [5], [11], [34]. Since these growth factors play an important role in normal brain function, it is of physiological significance to determine whether OPC responses to PDGF and bFGF are disrupted by Pb exposure in vitro.

PKC activation can affect OPC cell fate [4], [6], [34], and this enzyme has been implicated as a cellular target for Pb toxicity in various systems [13], [14], [23], [25], [39], [47]. Pb is known to activate PKC in the picomolar range [14], [28], and stimulation of PKC was found to delay oligodendrocyte differentiation [4], [6]. Accordingly, signal transduction pathways involving PKC may be important for Pb toxicity in oligodendrocyte progenitors.

The present study demonstrates that while Pb is capable of killing the cells, the metal can inhibit the proliferation and differentiation of oligodendrocyte lineage cells in vitro at concentrations that do not decrease cell viability. These responses to Pb exposure appear to require activation of PKC.

Section snippets

Culture and treatment of oligodendrocyte progenitors

Primary OPCs were isolated from mixed glial cultures of the forebrains of 2-day-old Sprague–Dawley rats using a selective detachment procedure, as described in detail elsewhere [16], [29]. The cultures were further purified by differential adhesion and trypsinization [18]. Progenitor cells were maintained in a chemically defined medium [30] with the addition of fresh recombinant human PDGF-AA (10 ng/ml) and bFGF (FGF-2, 5 ng/ml, Sigma) every 48 h. Cultures were routinely characterized by

Oligodendrocyte lineage cells can be cultured in minimal medium containing 1% FBS

Though a chemically defined medium has been used widely for the maintenance of OPCs [30], a minimal medium (DMEM) containing 1% FBS was employed in this study to minimize the effects of extraneous extracellular regulatory factors. We found that OPC growth rate and ability to differentiate into mature oligodendrocytes did not differ when cells were maintained (with PDGF and bFGF when appropriate) in minimal or defined medium (data not shown).

Pb is cytotoxic to oligodendrocyte lineage cells

The effect of Pb on cell viability was determined with

Discussion

Previous studies revealed that cultured oligodendroglial progenitors are highly vulnerable to Pb toxicity [16], [18] and may well be a significant target for environmental Pb exposure. The present study gives further insight into the Pb-mediated effects by determining the impact of the metal on OPC survival, proliferation and differentiation. Pb exposure for a period of 24 h is cytocidal to OPCs as evidenced by a loss of mitochondrial redox potential and activation of the apoptosis-related

Acknowledgements

We thank Sarah Owens and Josephine Incardona for assistance with the preparation of oligodendrocyte progenitor cultures, and Dr. Renping Zhou for his suggestions and critical review of this manuscript. This research was supported in part by grants to R.D.P. from NIEHS (R01-ES08373) and the March of Dimes Foundation.

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    Present address: Department of Neurology, Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA.

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