Regulation of astrocyte glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4) expression in a model of epilepsy

Abstract

Astrocytes regulate extracellular glutamate and water homeostasis through the astrocyte-specific membrane proteins glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4), respectively. The role of astrocytes and the regulation of GLT1 and AQP4 in epilepsy are not fully understood. In this study, we investigated the expression of GLT1 and AQP4 in the intrahippocampal kainic acid (IHKA) model of temporal lobe epilepsy (TLE). We used real-time polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analysis at 1, 4, 7, and 30days after kainic acid-induced status epilepticus (SE) to determine hippocampal glial fibrillary acidic protein (GFAP, a marker for reactive astrocytes), GLT1, and AQP4 expression changes during the development of epilepsy (epileptogenesis). Following IHKA, all mice had SE and progressive increases in GFAP immunoreactivity and GFAP protein expression out to 30days post-SE. A significant initial increase in dorsal hippocampal GLT1 immunoreactivity and protein levels were observed 1day post SE and followed by a marked downregulation at 4 and 7days post SE with a return to near control levels by 30days post SE. AQP4 dorsal hippocampal protein expression was significantly downregulated at 1day post SE and was followed by a gradual return to baseline levels with a significant increase in ipsilateral protein levels by 30days post SE. Transient increases in GFAP and AQP4 mRNA were also observed. Our findings suggest that specific molecular changes in astrocyte glutamate transporters and water channels occur during epileptogenesis in this model, and suggest the novel therapeutic strategy of restoring glutamate and water homeostasis.

Keywords: AQP4; Aquaporin; Astrocyte; EAAT2; Epilepsy; GFAP; GLT1; Glutamate transporter-1; Kainic acid; Seizure.

Fig. 1

Glial fibrillary acidic protein (GFAP) immunoreactivity in the ipsilateral hippocampus after kainic acid-induced status epilepticus (SE). A. 10× images of GFAP (red), DAPI (blue), and merged immunoreactivity of the ipsilateral hippocampus after saline injections (Saline control) and 1, 4, 7, and 30 days (d) post SE. B. Quantification of GFAP immunoreactivity in the various layers of the hippocampus. ** indicates p < 0.01 and *** indicates p < 0.001 when compared to saline control. Scale bar = 200 μm. SO = stratum oriens; SP = stratum pyramidale; SR = stratum radiatum; SLM = stratum lacunosum moleculare; ML = molecular layer; GCL = granule cell layer; H = hilus.

Fig. 1

Glial fibrillary acidic protein (GFAP) immunoreactivity in the ipsilateral hippocampus after kainic acid-induced status epilepticus (SE). A. 10× images of GFAP (red), DAPI (blue), and merged immunoreactivity of the ipsilateral hippocampus after saline injections (Saline control) and 1, 4, 7, and 30 days (d) post SE. B. Quantification of GFAP immunoreactivity in the various layers of the hippocampus. ** indicates p < 0.01 and *** indicates p < 0.001 when compared to saline control. Scale bar = 200 μm. SO = stratum oriens; SP = stratum pyramidale; SR = stratum radiatum; SLM = stratum lacunosum moleculare; ML = molecular layer; GCL = granule cell layer; H = hilus.

Fig. 2

Hippocampal protein and mRNA quantification of glial fibrillary acidic protein (GFAP) after kainic acid-induced status epilepticus (SE). A. Representative Western blot of GFAP (red) and β-actin (green) protein from the ipsilateral hippocampus of a saline control (Sal) and 1, 4, 7, and 30 days (d) after kainic acid-induced SE. B. Quantification of GFAP band intensities normalized to β-actin at each time point in the ipsilateral hippocampus. C. Fold change in GFAP mRNA levels in the ipsilateral hippocampal tissue from saline controls (Sal) and in tissue collected 1, 4, 7, and 30 days (d) post SE. D. Representative Western blot of GFAP (red) and β-actin (green) protein from the contralateral hippocampus from a saline control (Sal) and 1, 4, 7, and 30 days (d) post SE. E. Quantification of contralateral GFAP protein normalized to β-actin in each group. F. Quantification of GFAP mRNA levels in the contralateral hippocampus of saline controls (Sal) and various time points post SE. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 when compared to saline control and n = 5 for each time point.

Fig. 3

Glutamate transporter-1 (GLT1) immunoreactivity in the ipsilateral hippocampus after kainic acid-induced status epilepticus (SE). A. 10× images of GLT1 (green), DAPI (blue), and merged immunoreactivity of the ipsilateral hippocampus after saline injections (Saline control) and 1, 4, 7, and 30 days (d) post SE. B. Quantification of GLT1 immunoreactivity in the various layers of the hippocampus ipsilateral to kainic acid injections. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 when compared to saline control. Scale bar = 200 μm. SO = stratum oriens; SP = stratum pyramidale; SR = stratum radiatum; SLM = stratum lacunosum moleculare; ML = molecular layer; GCL = granule cell layer; H = hilus.

Fig. 3

Glutamate transporter-1 (GLT1) immunoreactivity in the ipsilateral hippocampus after kainic acid-induced status epilepticus (SE). A. 10× images of GLT1 (green), DAPI (blue), and merged immunoreactivity of the ipsilateral hippocampus after saline injections (Saline control) and 1, 4, 7, and 30 days (d) post SE. B. Quantification of GLT1 immunoreactivity in the various layers of the hippocampus ipsilateral to kainic acid injections. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 when compared to saline control. Scale bar = 200 μm. SO = stratum oriens; SP = stratum pyramidale; SR = stratum radiatum; SLM = stratum lacunosum moleculare; ML = molecular layer; GCL = granule cell layer; H = hilus.

Fig. 4

Glutamate transporter-1 (GLT1) immunoreactivity in the contralateral hippocampus after kainic acid-induced status epilepticus (SE). A. 10× images of GLT1 (green), DAPI (blue), and merged immunoreactivity of the contralateral hippocampus after saline injections (Saline control) and 1, 4, 7, and 30 days (d) post SE. B. Quantification of GLT1 immunoreactivity in the various layers of the hippocampus contralateral to kainic acid injections. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 when compared to saline control. Scale bar =200 μm. SO = stratum oriens; SP = stratum pyramidale; SR = stratum radiatum; SLM = stratum lacunosum moleculare; ML = molecular layer; GCL = granule cell layer; H = hilus.

Fig. 4

Glutamate transporter-1 (GLT1) immunoreactivity in the contralateral hippocampus after kainic acid-induced status epilepticus (SE). A. 10× images of GLT1 (green), DAPI (blue), and merged immunoreactivity of the contralateral hippocampus after saline injections (Saline control) and 1, 4, 7, and 30 days (d) post SE. B. Quantification of GLT1 immunoreactivity in the various layers of the hippocampus contralateral to kainic acid injections. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 when compared to saline control. Scale bar =200 μm. SO = stratum oriens; SP = stratum pyramidale; SR = stratum radiatum; SLM = stratum lacunosum moleculare; ML = molecular layer; GCL = granule cell layer; H = hilus.

Fig. 5

Dorsal hippocampal glutamate transporter-1 (GLT1) protein and mRNA levels after kainic acid-induced status epilepticus (SE). A. Representative Western blot of GLT1 (red) and β-actin (green) protein from the ipsilateral hippocampus of a saline control (Sal) and 1, 4, 7, and 30 days (d) post kainic acid-induced SE. B. Quantification of GLT1 lower band intensity normalized to β-actin at each time point in the ipsilateral hippocampus. C. Fold change in GLT1 mRNA in the ipsilateral hippocampus from tissue collected from saline controls (Sal) and at 1, 4, 7, and 30 days (d) post kainic acid-induced SE. D. Representative Western blot of GLT1 (red) and β-actin (green) protein from the contralateral hippocampus of a saline control (Sal) and 1, 4, 7, and 30 days post SE. E. Quantification of contralateral hippocampal GLT1 lower protein band normalized to β-actin in each group. F. Quantification of GLT1 mRNA levels in the contralateral hippocampus from tissue collected from saline controls (Sal) and at various time points post kainic acid-induced SE. * indicates p < 0.05 and ** indicates p < 0.01 when compared to saline control. For each time point, n = 5 except protein for 7 days post SE (n = 7).

Fig. 6

Dorsal hippocampal aquaporin-4 (AQP4) protein and mRNA levels after kainic acid-induced status epilepticus (SE). A. Representative Western blot of AQP4 (red) and β-actin (green) protein from the ipsilateral hippocampus from a saline control (Sal) and 1, 4, 7, and 30 days (d) post SE. B. Quantification of AQP4 band intensities normalized to β-actin at each time point from the ipsilateral hippocampus. C. Fold change in AQP4 mRNA in the ipsilateral hippocampus from saline controls (Sal) and 1, 4, 7, and 30 days (d) post SE. D. Representative Western blot of AQP4 (red) and β-actin (green) protein from the contralateral hippocampus in a saline control (Sal) and 1, 4, 7, and 30 days post kainic acid-induced SE. E. Quantification of AQP4 protein from the contralateral hippocampus normalized to β-actin in each group. F. Quantification of AQP4 mRNA levels in the contralateral hippocampus in tissue collected from saline controls (Sal) and at various time points post SE. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 when compared to saline control and n = 5 for each time point.