Research articleMonocarboxylate transporter-dependent mechanism confers resistance to oxygen- and glucose-deprivation injury in astrocyte-neuron co-cultures
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.
References (27)
- et al.
Highly differential expression of the monocarboxylate transporters MCT2 and MCT4 in the developing rat brain
Neuroscience
(2003) - et al.
Endogenous neuroprotection: mito-chondria as gateways to cerebral preconditioning
Neuropharmacology
(2008) - et al.
Ischemic tolerance and endogenous neuroprotection
Trends Neurosci.
(2003) - et al.
Monocarboxylate transporter 1 is deficient on microvessels in the human epileptogenic hippocampus
Neurobiol. Dis.
(2011) - et al.
Brain contains a functional glucose-6-phosphatase complex capable of endogenous glucose production
J. Biol. Chem.
(2005) - et al.
The plasma membrane lactate transporter MCT4 but not MCT1, is up-regulated by hypoxia through a HIF- 1alpha-dependent mechanism
J. Biol. Chem.
(2006) - et al.
Effect of lipoic acid and <alpha>-glyceryl-phosphoryl-choline on astroglial cell proliferation and differentiation in primary culture
J. Neurosci. Res.
(2014) - et al.
Effect of growth factors and steroid hormones on heme oxygenase and cyclin D1 expression in primary astroglial cell cultures
J. Neurosci. Res.
(2015) - et al.
Microglia-neuron interaction in inflammatory and degenerative diseases: role of cholinergic and noradrenergic systems
CNS Neurol. Disord. Drug Targets
(2007) - et al.
Cholinergic precursors modulate the expression of heme oxigenase-1, p21 during astroglial cell proliferation and differentiation in culture
Neurochem. Res.
(2012)
Glycogen metabolism and brain pathologies
Cent. Nerv. Syst. Agents Med. Chem.
Supply and demand in cerebral energy metabolism: the role of nutrient transporters
J. Cereb. Blood Flow Metab.
The SLC16 gene family-from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond
Pflugers Arch.
Cited by (25)
Lactate Supply from Astrocytes to Neurons and its Role in Ischemic Stroke-induced Neurodegeneration
2022, NeuroscienceCitation Excerpt :The expression of MCT is subtly regulated by various conditions. For example, glucose deficiency increases MCT4 expression in astrocytes (Gao et al., 2015). In case of blocked glucose supply to neuronal cells, neuronal cells cannot store glucose in glycogen form; therefore, neuronal cells are supplied with lactate from astrocytes to produce ATP (Brown and Ransom, 2007).
Astrocytic nutritional dysfunction associated with hypoxia-induced neuronal vulnerability in stroke-prone spontaneously hypertensive rats
2020, Neurochemistry InternationalCitation Excerpt :In addition, it was demonstrated that lactate may affect some homeostatic functions of neurons (Magistretti and Allaman, 2018). Thus, regulation of lactate supply from astrocytes to neurons via MCTs may be an important factor in conferring tolerance to nerve damage during oxygen and glucose deficiency (Gao et al., 2015). Stroke is caused by a decrease in cerebral blood flow due to cerebral ischemia.
Involvement of monocarboxylate transporters in the cross-tolerance between epilepsy and cerebral infarction: A promising choice towards new treatments
2019, Neuroscience LettersCitation Excerpt :Signals were detected using anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:5000; Abcam, Cambridge, MA, USA) and analyzed using Image J software. Immunofluorescence was performed as described previously [8]. Incubate antibodies as follows: antibodies against MCT1(Catalog #: sc-50325, 1:500; Santa Cruz Biotechnology, Inc.
Cell death in pure-neuronal and neuron-astrocyte mixed primary culture subjected to oxygen-glucose deprivation: The contribution of poly(ADP-ribose) polymerases and caspases
2018, Microchemical JournalCitation Excerpt :Moreover, depending on the OGD timing [30] and in combination with the mitochondrial damage [44], the caspase-9 mediated intrinsic apoptosis pathway is also involved in OGD-induced cell damage, leading to caspase-3 activation. A lactate-depending mechanism related to the glycogen storage confers astrocyte resistance to OGD [42,45]. We then evaluated if the presence of astrocytes in the culture affect neuron vulnerability to OGD.
- 1
Chen Gao and Liya Zhou Contributed equally to this work.