Elsevier

Neuroscience Letters

Volume 594, 6 May 2015, Pages 99-104
Neuroscience Letters

Research article
Monocarboxylate transporter-dependent mechanism confers resistance to oxygen- and glucose-deprivation injury in astrocyte-neuron co-cultures

https://doi.org/10.1016/j.neulet.2015.03.062Get rights and content

Highlights

  • 8 h OGD significantly increases cell death in primary neuronal cultures.

  • OGD up-regulates MCT4 expression in primary astrocyte cultures.

  • Neuronal cell death in co-cultures is increased by exposure to MCTs-specific siRNA under OGD.

  • Exogenous lactate in the extracellular medium can protect neuronal cultures from OGD.

Abstract

Hypoxic and low-glucose stressors contribute to neuronal death in many brain diseases. Astrocytes are anatomically well-positioned to shield neurons from hypoxic injury. During hypoxia/ischemia, lactate released from astrocytes is taken up by neurons and stored for energy. This process is mediated by monocarboxylate transporters (MCTs) in the central nervous system. In the present study, we investigated the ability of astrocytes to protect neurons from oxygen- and glucose-deprivation (OGD) injury via an MCT-dependent mechanism in vitro. Primary cultures of neurons, astrocytes, and astrocytes–neurons derived from rat hippocampus were subjected to OGD, MCT inhibition with small interfering (si)RNA. Cell survival and expression of MCT4, MCT2, glial fibrillary acidic protein, and neuronal nuclear antigen were evaluated. OGD significantly increased cell death in neuronal cultures and up-regulated MCT4 expression in astrocyte cultures, but no increased cell death was observed in neuron–astrocyte co-cultures or astrocyte cultures. However, neuronal cell death in co-cultures was increased by exposure to MCT4- or MCT2-specific siRNA, and this effect was attenuated by the addition of lactate into the extracellular medium of neuronal cultures prior to OGD. These findings demonstrate that resistance to OGD injury in astrocyte–neuron co-cultures occurs via an MCT-dependent mechanism.

Introduction

Astrocytes are involved in the physical structuring of the brain. They are the most abundant glial cells in the brain that are closely associated with neuronal synapses [1]. Glial cells are also involved in providing neurotrophic signals to neurons required for their survival, proliferation, and differentiation [2]. In addition, reciprocal interactions between glia and neurons are essential for many critical functions in brain health and disease. Glial cells play pivotal roles in neuronal development, activity, plasticity, and recovery from injury [3]. The idea that astrocytes have active roles in the modulation of neuronal activity and synaptic neurotransmission is now widely accepted [4]. Lactate released from astrocytes via glycogenolysis and glycolysis is taken up by neurons and used for energy [5]. Monocarboxylate transporters (MCTs), which are abundantly expressed in neurons and astrocytes, play an important role in this process [6]. MCTs belong to the SLC16 gene family, which comprises 14 members. MCT1–4 are proton symporters that mediate the transmembrane transport of lactate, pyruvate, and ketone bodies [7]; MCT2 is expressed primarily in neurons in the brain, while MCT4 is expressed almost exclusively in astrocytes [8]. During hypoxia/ischemia, lactate released from astrocytes is taken up by neurons and stored for energy via up-regulation of MCT4 expression [9]. Glial fibrillary acidic protein (GFAP) is an astrocyte-differentiation marker and is considered to be an important element in astrocyte differentiation and in the reactive response to central nervous system injury [10].

In the present study, we investigated the ability of astrocytes to protect neurons from oxygen- and glucose-deprivation (OGD) injury via an MCT-dependent mechanism in vitro. Primary neuronal, astrocyte, and astrocyte–neuron co-cultures derived from rat hippocampus were subjected to OGD, and MCT4, MCT2, GFAP and neuronal nuclear antigen (NeuN) expression levels were evaluated.

Section snippets

Rat primary astrocyte–neuron co-cultures

All procedures were approved by the Animal Care and Use Committee of LanZhou University (Lanzhou, China) and followed the National Guidelines for Animal Experimentation. Hippocampuses were obtained from 18-day Sprague–Dawley rat embryos (n = 7), using a modification of a previously-described method [11]. The cells were plated on culture flasks or glass coverslips in a six-well plate at around 1–2×105 cells/cm2 and maintained in Neurobasal-A growth medium without fetal bovine serum (FBS) at 37 °C

Effect of OGD on cell survival

We identified the effects of OGD on survival of different primary cell cultures using a live/dead cell assay (Fig. 1A and B), with cells cultured under normal conditions as controls. Compared with control cells, OGD significantly increased cell death in neuronal cultures (65.6%), but not in co-cultures and astrocyte cultures. Apoptosis was determined by nuclear staining with DAPI. Following 8 h of OGD, 64.2% of neurons in neuronal cultures were apoptotic, compared with 6.5% in control cells (

Discussion

Increased glycogen stores in cultured astrocytes have been shown to protect neurons from ischemia and glucose deprivation [14]. Astrocytes are thought to play a major role in supplying neurons with energy in the form of lactate during periods of intense neural activity, when their energy demands exceed the supply of glucose from the blood, as suggested by the astrocyte–neuron lactate shuttle hypothesis (ANLSH) [15]. Lactate is, thus, emerging as a potential neuroprotective agent, as well as a

Acknowledgments

The authors thank Dafu Zhang for help with the RT-PCR protocol and Xiangyang Wang for his contributions to this work.

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    Chen Gao and Liya Zhou Contributed equally to this work.

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