Conditional Deletion of the Glutamate Transporter GLT-1 Reveals That Astrocytic GLT-1 Protects against Fatal Epilepsy While Neuronal GLT-1 Contributes Significantly to Glutamate Uptake into Synaptosomes

Abstract

GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAPCreERT2. Elimination of GLT-1 from astrocytes resulted in loss of ϳ80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (V max ) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake.

Neuronal vs glial glutamate uptake: Resolving the conundrum

“…The mice appeared normal with normal lifespan. From this it was concluded that astroglial EAAT2 is the main protector against excitotoxicity (Petr et al, 2015) in agreement with the distribution of the EAAT2 protein.…”

“…In agreement, it was shown electron microscopically that D-aspartate accumulates preferentially (85 %) in the terminals in synaptosome preparations (Furness et al, 2008). This was tested using conditional knockout mice: when EAAT2 was selectively deleted in neurons (using synapsin-Cre mice), then the uptake activity in synaptosome preparations was reduced to half (Petr et al, 2015; see also Fig. 4).…”

“…Three different research teams generated, independently of each other, their own flox-EAAT2 mice (Zhou et al, 2014b;Petr et al, 2015;Aida et al, 2015). The first of these three mouse lines (Zhou et al, 2014b) is already available from Jackson Laboratory (JAX Stock No.…”

“…Thus, Cre-mediated expression in neurons can be avoided by injecting tamoxifen at postnatal day 5 or later. This resulted in mice where the levels of EAAT2 protein in the whole forebrain were reduced to 1/5 of the normal level resulting in spontaneous epilepsy and increased mortality (Petr et al, 2015) albeit not quite as dramatic as the global knockouts. In contrast, deletion in neurons by crossing the flox-EAAT2 mice with synapsin I promoter-driven Cre recombinase mice (Rempe et al, 2006; Jackson Laboratory: JAX Stock No.…”

Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes

“…For immunoblot analysis, astrocytes or striatal tissue were lysed by frosting and defrosting in lysis buffer (containing Tris HCl 0.2 M, pH 8, and Protease Inhibitor 1ϫ, Sigma) and centrifuged at 10,000 ϫ g for 10 min at 4°C as described previously (Petr et al, 2015): mouse anti-␤-actin (1:15000; Santa Cruz Biotechnologies); mouse anti-EAAT2/GLT-1 (1: 1000; Millipore clone G6); and rabbit anti-EAAT1/GLAST (1:500; Abcam). Western blots were quantified measuring optical densities of immunoreactive protein bands (in vitro only monomer of GLT-1 was present) using ImageJ or Image Laboratory (Bio-Rad Laboratories) software, normalizing results to ␤-actin values.…”