Elsevier

Neuroscience

Volume 155, Issue 4, 9 September 2008, Pages 1204-1211
Neuroscience

Molecular neuroscience
Selective overexpression of excitatory amino acid transporter 2 (EAAT2) in astrocytes enhances neuroprotection from moderate but not severe hypoxia–ischemia

https://doi.org/10.1016/j.neuroscience.2008.05.059Get rights and content

Abstract

Attempts have been made to elevate excitatory amino acid transporter 2 (EAAT2) expression in an effort to compensate for loss of function and expression associated with disease or pathology. Increased EAAT2 expression has been noted following treatment with β-lactam antibiotics, and during ischemic preconditioning (IPC). However, both of these conditions induce multiple changes in addition to alterations in EAAT2 expression that could potentially contribute to neuroprotection. Therefore, the aim of this study was to selectively overexpress EAAT2 in astrocytes and characterize the cell type specific contribution of this transporter to neuroprotection. To accomplish this we used a recombinant adeno-associated virus vector, AAV1–glial fibrillary acidic protein (GFAP)–EAAT2, designed to selectively drive the overexpression of EAAT2 within astrocytes. Both viral-mediated gene delivery and β-lactam antibiotic (penicillin-G) treatment of rat hippocampal slice cultures resulted in a significant increase in both the expression of EAAT2, and dihydrokainate (DHK) sensitive glutamate uptake. Penicillin-G provided significant neuroprotection in rat hippocampal slice cultures under conditions of both moderate and severe oxygen glucose deprivation (OGD). In contrast, viral-mediated overexpression of EAAT2 in astrocytes provided enhanced neuroprotection only following a moderate OGD insult. These results indicate that functional EAAT2 can be selectively overexpressed in astrocytes, leading to enhanced neuroprotection. However, this cell type specific increase in EAAT2 expression offers only limited protection compared to treatment with penicillin-G.

Section snippets

Virus preparation

The AAV1-GFAP-eGFP and AAV1-GFAP-EAAT2 viruses were packaged in HEK293T cells cultures grown in standard growth media (DMEM, 10% heat inactivated FBS, 0.05% penicillin/streptomycin (5000 U/ml), 0.1 mM MEM nonessential amino acids, 1 mM MEM sodium pyruvate, and gentamicin (25 mg/ml). Cells were transfected with three plasmids using Polyfect Transfection Reagent (Qiagen, Valencia, CA, USA). The three plasmids used in the transfection were: 1) adeno helper plasmid (pFΔ6), AAV helper (H21) and the

Astrocyte-targeted transgene expression following AAV-mediated gene delivery

The GFAP promoter sequence identified by Brenner et al. (1994) has been used in multiple studies to selectively drive protein expression in astrocytes. Feng et al. (2004) demonstrated stable, long-term astrocyte specific expression of apolipoprotein E (ApoE) using rAAV containing the GFAP promoter. Guo et al. (2003) utilized the GFAP promoter sequence to drive EAAT2 expression in a transgenic mouse model and noted a high degree of astrocyte specific transgene expression.

We validated the

Discussion

Prior attempts to modulate EAAT2 function have been limited to non-specific or indirect approaches. This is due to the fact that only a limited number of inhibitory compounds exhibit transporter isotype specificity. In addition, all currently available inhibitors are unable to target transporters in a cell type specific manner. Likewise, enhancers of EAAT2 function, such as the β-lactam antibiotics, also lack cell type specificity.

In an effort to circumvent this limitation, Guo et al. (2003)

Acknowledgments

This publication was supported by grants from NCRR and NINDS (P20 RR15583, P20 RR017670, R21 NS058541-01).

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    Present address: NIH/NIDCR, Building 10, Magnuson CC, 1N103, 10 Center Drive, Bethesda, MD, USA; E-mail address: [email protected].

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