Nuclear Factor- B Contributes to Neuron-Dependent Induction of Glutamate Transporter-1 Expression in Astrocytes

Abstract

The GLT-1 (EAAT2) subtype of glutamate transporter ensures crisp excitatory signaling and limits excitotoxicity in the CNS. Astrocytic expression of GLT-1 is regulated during development, by neuronal activity, and in neurodegenerative diseases. Although neurons activate astrocytic expression of GLT-1, the mechanisms involved have not been identified. In the present study, astrocytes from transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of a bacterial artificial chromosome (BAC) containing a very large region of DNA surrounding the GLT-1 gene (BAC GLT-1 eGFP mice) were used to assess the role of nuclear factor-κB (NF-κB) in neuron-dependent activation of the GLT-1 promoter. We provide evidence that neurons activate NF-κB signaling in astrocytes. Transduction of astrocytes from the BAC GLT-1 eGFP mice with dominant-negative inhibitors of NF-κB signaling completely blocked neuron-dependent activation of a NF-κB reporter construct and attenuated induction of eGFP. Exogenous expression of p65 and/or p50 NF-κB subunits induced expression of eGFP or GLT-1 and increased GLT-1-mediated transport activity. Using wild type and mutant GLT-1 promoter reporter constructs, we found that NF-κB sites at −583 or −251 relative to the transcription start site eliminated neuron-dependent reporter activation. Electrophoretic mobility shift and supershift assays reveal that p65 and p50 interact with these same sites ex vivo. Finally, chromatin immunoprecipitation (ChIP) showed that p65 and p50 interact with these sites in adult cortex, but not in kidney (a tissue that expresses no detectable GLT-1). Together, these studies strongly suggest that NF-κB contributes to neuron-dependent regulation of astrocytic GLT-1 transcription.

Riluzole effects on behavioral sensitivity and the development of axonal damage and spinal modifications that occur after painful nerve root compression

“…11,35,38,69 GLT-1 is only expressed by glial cells in the rat spinal cord, 70 and astrocytes require neuronal signaling to express GLT-1. 6,23,77 The downregulation of GLT-1 after a painful nerve root compression may be due to impaired afferent neurotransmission in the superficial laminae that is associated with the overall decrease in neuropeptides in this region (Figs. 3 and 4).…”

“…2 and 4). 5,23,55,76,77 Downregulation of GLT-1 expression in the superficial laminae suggests that glutamate uptake is impaired (Fig. 4), 11,70 which has been associated with increased spinal glutamate concentration, 11,35,38 and may contribute to the increase in the amplitude of excitatory currents on postsynaptic neurons in the superficial laminae that are reported after a sustained painful root constriction.…”

Genetic Dys-regulation of Astrocytic Glutamate Transporter EAAT2 and its Implications in Neurological Disorders and Manganese Toxicity

“…Moreover, neurons enhance EAAT2 expression in astrocytes when they are co-cultured [47, 48]. Although factors released from neurons that are responsible for increasing astrocytic EAAT2 expression are not well understood, NF-κB appears to be critically involved in neuronal activation of EAAT2 [49]. NF-κB binding sites at −583 or −251 of the EAAT2 promoter are important for neuronal activation of EAAT2 promoter activity and both NF-κB isoforms, p65 and p50, interact with these sites to enhance EAAT2 promoter activity [49].…”

“…Although factors released from neurons that are responsible for increasing astrocytic EAAT2 expression are not well understood, NF-κB appears to be critically involved in neuronal activation of EAAT2 [49]. NF-κB binding sites at −583 or −251 of the EAAT2 promoter are important for neuronal activation of EAAT2 promoter activity and both NF-κB isoforms, p65 and p50, interact with these sites to enhance EAAT2 promoter activity [49]. In addition, the activation of kappa B-motif binding phospho-protein (KBPP) is involved in neuronal activation of EAAT2 promoter activity [50].…”

GLT-1 transporter: An effective pharmacological target for various neurological disorders

“…The effect of TX on astrocytic GLT-1/EAAT-2 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway (Karki et al, 2013). In addition, the effect of TX on GLT-1/EAAT-2 promoter activity was abolished by the inhibition of the NF-κB pathway (Ghosh et al, 2011). Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1/EAAT-2 promoter.…”