FK506 suppresses hippocampal seizures and neuroinjury by upregulating astrocytic EAAT1/2 expression via inhibition of calcineurin activity

Calcineurin (CaN) is implicated in regulating hippocampal astrocytic excitatory amino acid transporter 1 (EAAT1) and 2 (EAAT2) expression. This study investigates whether tacrolimus (FK506) reduces epileptogenesis by upregulating EAAT1 and EAAT2 in hippocampal astrocytes in a kainic acid (KA)-induced epilepsy model.

The cellular distribution of EAAT1, EAAT2, CaN, GFAP, and NeuN was analyzed using human and mouse brain single cell RNA sequencing (scRNA seq) datasets (GSE190453, GSE241349) from the GEO database. Hippocampal astrocyte RNA seq data from KA-induced epileptic mice (GSE237321) were used to examine the expression patterns of EAAT1, EAAT2, CaN, and GFAP. A total of 108 specific pathogen free (SPF) C57 BL/6J mice aged 6 to 8 weeks (body weight 19 to 23 g, equal numbers of males and females) were used. To evaluate the effect of low dose FK506 on cortical discharges and the regulation of EAAT1 and EAAT2, 12 mice were randomly divided into Control, KA, and FK506 groups (n=4). The FK506 group received a single intraperitoneal injection of 1 mg/kg FK506 24 h and 1 h before modeling; the KA group was modeled by bilateral hippocampal injection of 0.5 μL KA (0.5 μg/μL). Cortical discharges and hippocampal EAAT1 and EAAT2 mRNA levels were assessed 48 h after KA injection. To determine the temporal expression profiles of EAAT1, EAAT2, CaN, and GFAP proteins and identify appropriate sampling time points, 36 mice were randomly allocated to Control, 6 h, 1 d, 3 d, 5 d, and 7 d groups (n=6). Hippocampal tissues were collected at the corresponding time points after KA administration for Western blotting. To evaluate the efficacy of different FK506 doses, 60 mice were divided n into Control, KA, FK506 1 mg/kg, FK506 2 mg/kg, and FK506 4 mg/kg groups (n=10). FK506 treatment groups received corresponding doses (intraperitoneal injection) 24 h and 1 h before KA modeling, followed by once daily injections for 3 consecutive days after modeling. On day 5 after modeling, open field testing was performed to assess locomotor activity. Hippocampal tissues from 6 mice per group were used for Western blotting of EAAT1, EAAT2, CaN, and GFAP; the remaining mice were used for HE staining to observe neuronal pathology and immunofluorescence staining to examine the colocalization of EAAT1 and EAAT2 with GFAP.

scRNA-seq analysis revealed that EAAT1 and EAAT2 were specifically localized in astrocytes, and CaN was also expressed in astrocytes. Hippocampal astrocyte RNA seq data showed that compared with PBS and AAV groups, EAAT1 and EAAT2 mRNA levels were significantly downregulated 4 d after KA induction, while GFAP and CaN were significantly upregulated. Immunofluorescence of mouse hippocampal sections indicated that EAAT1 and EAAT2 co-localized with the astrocyte marker GFAP, and GFAP fluorescence intensity was significantly increased in the KA group versus the Control group. Western blotting demonstrated that EAAT1 and EAAT2 were significantly downregulated in the 5 d group compared with the Control group; GFAP levels began to rise 6 h after KA treatment and were significantly increased at 3 d; full length CaN protein levels decreased from 3 d onward, whereas the 45 kDa CaN active fragment was significantly increased at 3 d and 5 d, suggesting that CaN is activated early in epileptogenesis and may participate in regulating EAAT1 and EAAT2 protein expression. Pretreatment with 1 mg/kg FK506 ameliorated abnormal cortical discharges in KA mice and partially alleviated KA induced downregulation of EAAT1 and EAAT2 mRNA. Continuous post modeling intervention with different doses of FK506 reduced neuronal damage in hippocampal CA1 and CA3 regions and improved motor deficits. Western blotting showed that different doses of FK506 inhibited KA-induced downregulation of EAAT1 and EAAT2 expression, suppressed the decrease in full length CaN and the generation of the 45 kDa CaN active fragment, partially inhibited GFAP upregulation, and significantly mitigated NeuN downregulation, indicating that FK506 exerts neuroprotective effects in a dose dependent manner.

In the KA induced epileptic mouse model, FK506 upregulates the expression of EAAT1 and EAAT2 in hippocampal astrocytes by inhibiting the production of the CaN active fragment, thereby attenuating epilepsy-induced neuronal damage.