Elsevier

Neuropharmacology

Volume 58, Issue 2, February 2010, Pages 551-558
Neuropharmacology

Cannabinoid receptor activation reduces TNFα-Induced surface localization of AMPAR-type glutamate receptors and excitotoxicity

https://doi.org/10.1016/j.neuropharm.2009.07.035Get rights and content

Abstract

After injury or during neurodegenerative disease in the central nervous system (CNS), the concentration of tumor necrosis factor alpha (TNFα) rises above normal during the inflammatory response. In vitro and in vivo, addition of exogenous TNFα to neurons has been shown to induce rapid plasma membrane-delivery of AMPA-type glutamate receptors (AMPARs) potentiating glutamatergic excitotoxicity. Thus the discovery of drug targets reducing excess TNFα-induced AMPAR surface expression may help protect neurons after injury. In this study, we investigate the neuroprotective role of the CB1 cannabinoid receptor using quantitative immunofluorescent and real-time video microscopy to measure the steady-state plasma membrane AMPAR distribution and rate of AMPAR exocytosis after TNFα exposure in the presence or absence of CB1 agonists. The neuroprotective potential of CB1 activation with TNFα was measured in hippocampal neuron cultures challenged by an in vitro kainate (KA)-mediated model of Excitotoxic Neuroinflammatory Death (END). Here, we demonstrate that CB1 activation blocks the TNFα-induced increase in surface AMPARs and protects neurons from END. Thus, neuroprotective strategies which increase CB1 activity may help to reduce the END that occurs as a result of a majority of CNS insults.

Introduction

Control over normal neuronal glutamatergic signaling depends largely on the localization of AMPARs on synaptic and extrasynaptic plasma membrane (Malinow and Malenka, 2002). This balance can be disrupted during CNS injury or disease when the inflammatory response is initiated and the population of AMPARs on neuronal plasma membrane increases dramatically, potentiating END (reviewed in Leonoudakis et al., 2004, Pickering et al., 2005). We previously characterized TNFα as a powerful glial-derived instigator of AMPAR trafficking to the plasma membrane (Beattie et al., 2002, Stellwagen et al., 2005). The presence of TNFα in the CNS is required for synaptic development (Stellwagen and Malenka, 2006), but the increased TNFα concentrations induced following infection or injury (Nemeth et al., 1997) contribute to END (Lock et al., 1999, Nagatsu et al., 2000, New et al., 1998, Perry et al., 2001, Shohami et al., 1999, Szelenyi, 2001). Several groups attribute this toxicity to long-term, translation-dependent apoptotic signaling pathways (Fontaine et al., 2002, Reimann-Philipp et al., 2001, Yang et al., 2002, Zhao et al., 2001, Zou and Crews, 2005). However, TNFα also induces a rapid (within 15 min) increase in surface expression of AMPARs (Beattie et al., 2002, Ogoshi et al., 2005, Stellwagen et al., 2005) which are calcium permeable and principally localized extrasynaptically (Leonoudakis et al., 2008). Additionally, acute neurotrauma increases surface expression of calcium permeable AMPARs composed of GluR1 subunits (Grooms et al., 2000, Grossman et al., 1999, Sanchez et al., 2001, Ying et al., 1998), and potentiates neuronal excitotoxicity (Feldmeyer et al., 1999, Oguro et al., 1999). Together, these studies suggest that TNFα-induced AMPAR surface expression may be a mechanism contributing to the early stages of END. Agents which decrease TNFα-induced AMPAR surface expression may reduce excitotoxicity in neurons and our previous studies suggest the use of cannabinoid receptor agonists may serve as a potential pharmacological intervention (Abood et al., 2001).

The cannabinoid receptor system is found in hippocampal neurons (Leterrier et al., 2006, Mackie, 2007, McDonald et al., 2007) and protects against excitotoxicity (Kim et al., 2006, Marsicano et al., 2003, Shen and Thayer, 1998, Shen and Thayer, 1999). Activation of the G-protein-coupled CB1 cannabinoid receptor initiates complex signaling cascades and leads to the hyperpolarization of neuronal membranes by increasing potassium and decreasing calcium conductance (Howlett, 2002). Our current results suggest a novel neuroprotective mechanism under the control of CB1, namely the reduction of excessive AMPAR surface expression caused by TNFα. We have used hippocampal culture systems to examine AMPAR surface expression and neuron survival after KA-induced excitotoxicity while exogenously applying TNFα. Our results demonstrate that CB1 activation prevents the TNFα-induced increase in surface GluR1-containing AMPARs and protects neurons from TNFα potentiated excitotoxic stress.

Section snippets

Preparation of hippocampal cultures

Mixed hippocampal neuron cultures were prepared from E18 rat pups as previously described (Beattie et al., 2002). Briefly, hippocampi of embryonic day 18 (E18) Sprague Dawley rats were removed, digested with papain, and dissociated by trituration. Neurons were plated in Neurobasal medium containing B27 supplement and Glutamax (Invitrogen) on coverslips or plastic plates coated with poly-d-lysine. After cell attachment (3–4 h), the medium was replaced. Three days later, an equal volume of

Expression of GluR1 and CB1 receptors on hippocampal neurons

In hippocampal neurons, GluR1-containing AMPARs colocalize with postsynaptic markers on somatic and dendritic plasma membranes (Beattie et al., 2002, Stellwagen et al., 2005) and CB1 has been detected in plasma membrane and cytosolic compartments of somato-dendritic regions as well as on axonal plasma membranes (Leterrier et al., 2006, McDonald et al., 2007). To confirm surface expression of CB1 in or near postsynaptic membrane in our hippocampal neuron cultures we have used immunofluorescence

Discussion

Our data is supportive of the hypothesis that activation of CB1 decreases TNFα-induced surface expression of AMPARs and protects against END in hippocampal cultures. CB1, AMPARs, and TNFα receptors are all expressed in dendrites in our cultured hippocampal neurons where intracellular signaling between CB1 and TNFα receptors may compete to regulate AMPAR trafficking (Marsicano et al., 2003) as suggested in studies of cortical pyramidal neurons (Hill et al., 2007, Kim et al., 2006) and spinal

Acknowledgments

MEA is supported by grants from the NIH (DA09978, DA05274) and by The Forbes Norris ALS Center. EB is supported by grants from the NIH (MH067931 and NS038079) as well as by The Forbes Norris ALS Center, the California Pacific Medical Center Research Institute, and ALSA (starter grant 766). We thank Robert Kim and Sean Prasad for excellent technical assistance with the live imaging experiments. We thank Dr. Patricia Reggio (University of North Carolina, Greensboro) and Dr. Herb Seltzman (RTI)

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