Increased adenine nucleotide translocator 1 in reactive astrocytes facilitates glutamate transport

Abstract

A hallmark of central nervous system (CNS) pathology is reactive astrocyte production of the chronic glial scar that is inhibitory to neuronal regeneration. The reactive astrocyte response is complex; these cells also produce neurotrophic factors and are responsible for removal of extracellular glutamate, the excitatory neurotransmitter that rises to neurotoxic levels in injury and disease. To identify genes expressed by reactive astrocytes, we employed an in vivo model of the glial scar and differential display PCR and found an increase in the level of Ant1, a mitochondrial ATP/ADP exchanger that facilitates the flux of ATP out of the mitochondria. Ant1 expression in reactive astrocytes is regulated by transforming growth factor-β1, a pluripotent CNS injury-induced cytokine. The significance of increased Ant1 is evident from the observation that glutamate uptake is significantly decreased in astrocytes from Ant1 null mutant mice while a specific Ant inhibitor reduces glutamate uptake in wild-type astrocytes. Thus, the astrocytic response to CNS injury includes an apparent increase in energy mobilization capacity by Ant1 that contributes to neuroprotective, energy-dependent glutamate uptake.

Introduction

Reactive astrogliosis describes the response of astrocytes to central nervous system (CNS) injury. This response is defined by a dramatic increase in the number and size of glial fibrillary acidic protein (GFAP)-positive astrocytes and is one of the most consistent findings in neurologic disease and after traumatic CNS injury. Despite the prevalence of this response, the functional significance of reactive gliosis is poorly understood. Reactive astrocytes contribute to axonal regenerative failure at the site of injury by forming the glial scar, a dense network of processes and growth inhibitory extracellular matrix (ECM) molecules Bovolenta et al 1997, McKeon et al 1995. These cells also protect vulnerable neurons after injury by maintaining ion homeostasis in the brain (Takahashi et al., 1997) and by producing neurotrophic factors that support neuronal survival (Rudge et al., 1992). Astrocytes contribute to termination of excitatory synaptic activity by removing the neurotransmitter glutamate from the extracellular space via astrocyte-specific glutamate transporters Lehre et al 1995, Rothstein et al 1996, Tanaka et al 1997. Glutamate uptake is of particular importance following CNS injury because excessive glutamate released by damaged neurons can initiate a self-propagating excitotoxic cascade beginning with the overstimulation of glutamate receptors, excessive Ca²⁺ influx, oxidative stress, and ultimately neuronal cell death with concomitant additional glutamate release (Greene and Greenamyre, 1996). The capacity for glutamate uptake may be overwhelmed and glutamate excitotoxicity perpetuated Eng et al 1997, Ye and Sontheimer 1998. The multifaceted nature of the astrocytic response to injury is evident following ablation of reactive astrocytes in ganciclovir-treated transgenic mice expressing the herpes simplex thymidine kinase gene under the control of the GFAP promoter. Exacerbated neuronal cell death in these mice following cerebral cortex stab wound injury was reduced by an NMDA glutamate receptor antagonist, indicating elevated (excitotoxic) extracellular glutamate levels in the absence of astrocytes. Moreover, these animals display increased neurite outgrowth throughout the injured area from intact neurons surrounding the lesion (Bush et al., 1999). Thus, reactive astrocytes play a role in both neuroprotection and inhibition of neurite outgrowth after CNS injury.

Several cytokines and growth factors that regulate the glial response to injury are elevated in areas of CNS damage Lindholm et al 1992, Logan et al 1992, Mocchetti et al 1996, Wang et al 1997a. TGF-β1 is a particularly potent stimulator of reactive astrogliosis. Astrocytes exposed to TGF-β1 increase expression of GFAP Krohn et al 1999, Reilly et al 1998 as well as putative axon growth inhibitory ECM proteins such as tenascin and neurocan Asher et al 2000, Smith and Hale 1997. Intracerebral cortical injection of TGF-β1 increases astrocytic production of the ECM molecules laminin and fibronectin in areas of reactive gliosis, and in vivo administration of TGF-β neutralizing antibodies reduces ECM deposition following brain injury Logan et al 1994, Logan et al 1999

The molecular mechanisms of reactive gliosis, including the role of TGF-β1 in this process, are largely unexplored. Therefore, we employed differential display PCR (ddPCR) to identify genes specifically regulated by TGF-β1 in an in vivo model of reactive gliosis. We demonstrate that mRNA levels for the adenine nucleotide translocator isoform 1 (Ant1) are elevated in areas of reactive gliosis and regulated by TGF-β. Glutamate transport by Ant1^−/− astrocytes is reduced by 70%, indicating an important role for Ant1 in this process. These data suggest that mobilization of mitochondrial ATP may be a key component of the astrocytic response to CNS injury.