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GLT-1: The elusive presynaptic glutamate transporter.

ABSTRACT: Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiological significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (Vmax) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5-10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate homeostasis associated with normal functions, neurodegeneration, and response to drugs.

SUBMITTER: Rimmele TS

PROVIDER: S-EPMC5070539 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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GLT-1: The elusive presynaptic glutamate transporter.

Rimmele Theresa S TS Rosenberg Paul A PA

Neurochemistry international 20160426

Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Her ...[more]

PMID: 27129805

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Project description:Neuron-secreted factors induce astrocytic expression of the glutamate transporter, GLT-1 (excitatory amino acid transporter 2). In addition to their elaborate anatomic relationships with neurons, astrocytes also have processes that extend to and envelop the vasculature. Although previous studies have demonstrated that brain endothelia contribute to astrocyte differentiation and maturation, the effects of brain endothelia on astrocytic expression of GLT-1 have not been examined. In this study, we tested the hypothesis that endothelia induce expression of GLT-1 by co-culturing astrocytes from mice that utilize non-coding elements of the GLT-1 gene to control expression of reporter proteins with the mouse endothelial cell line, bEND.3. We found that endothelia increased steady state levels of reporter and GLT-1 mRNA/protein. Co-culturing with primary rat brain endothelia also increases reporter protein, GLT-1 protein, and GLT-1-mediated glutamate uptake. The Janus kinase/signal transducer and activator of transcription 3, bone morphogenic protein/transforming growth factor β, and nitric oxide pathways have been implicated in endothelia-to-astrocyte signaling; we provide multiple lines of evidence that none of these pathways mediate the effects of endothelia on astrocytic GLT-1 expression. Using transwells with a semi-permeable membrane, we demonstrate that the effects of the bEND.3 cell line are dependent upon contact. Notch has also been implicated in endothelia-astrocyte signaling in vitro and in vivo. The first step of Notch signaling requires cleavage of Notch intracellular domain by γ-secretase. We demonstrate that the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester blocks endothelia-induced increases in GLT-1. We show that the levels of Notch intracellular domain are higher in nuclei of astrocytes co-cultured with endothelia, an effect also blocked by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Finally, infection of co-cultures with shRNA directed against recombination signal binding protein for immunoglobulin kappa J, a Notch effector, also reduces endothelia-dependent increases in enhanced green fluorescent protein and GLT-1. Together, these studies support a novel role for Notch in endothelia-dependent induction of GLT-1 expression. Cover Image for this issue: doi. 10.1111/jnc.13825.

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GLT-1: The elusive presynaptic glutamate transporter.

Publications

GLT-1: The elusive presynaptic glutamate transporter.

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