Contrasting patterns of Bim induction and neuroprotection in Bim-deficient mice between hippocampus and neocortex after status epilepticus

Abstract

Prolonged seizures (status epilepticus) are associated with brain region-specific regulation of apoptosis-associated signaling pathways. Bcl-2 homology domain 3-only (BH3) members of the Bcl-2 gene family are of interest as possible initiators of mitochondrial dysfunction and release of apoptogenic molecules after seizures. Previously, we showed that expression of the BH3-only protein, Bcl-2 interacting mediator of cell death (Bim), increased in the rat hippocampus but not in the neocortex after focal-onset status epilepticus. In this study, we examined Bim expression in mice and compared seizure damage between wild-type and Bim-deficient animals. Status epilepticus induced by intra-amygdala kainic acid (KA) caused extensive neuronal death within the ipsilateral hippocampal CA3 region. Hippocampal activation of factors associated with transcriptional and posttranslational activation of Bim, such as CHOP and c-Jun NH(2)-terminal kinases, was significant within 1 h. Upregulation of bim mRNA was evident after 2 h and Bim protein increased between 4 and 24 h. Hippocampal CA3 neurodegeneration was reduced in Bim-deficient mice compared with wild-type animals after seizures in vivo, and short interfering RNA molecules targeting bim reduced cell death after KA treatment of hippocampal organotypic cultures. In contrast, neocortical Bim expression declined after status epilepticus, and neocortex damage in Bim-deficient mice was comparable with that in wild-type animals. These results show region-specific differential contributions of Bim to seizure-induced neuronal death.

Figure 1. Upregulation of Bim in the hippocampus during seizure-induced neuronal death

(A) Traces of two high amplitude high frequency seizure burst events captured using combined intra-hippocampal-cortical EEG. Arrows demark point of onset. Note that seizure activity is detected in the hippocampus (Hip) before the cortex (Ctx). (B) Photomicrographs (4× lens) show representative Fluoro-Jade B staining at 24 h in hippocampus of control mice and mice that underwent status epilepticus. Arrows denote cells (white) undergoing degeneration. (C) Graph showing quantification of mRNA levels for bim corrected to β-actin in control (C, 2 h) and seizure-damaged mice at various time points after lorazepam was given. Data are from four independent experiments. (D) Representative Western blot (n = 2 per lane) showing increased expression of Bim from 4–24 h after seizures compared to control and 1 h post-seizure. Actin is shown as a control for protein loading. (E) Graph showing semi-quantification of Bim protein levels. Data are from three independent experiments. A.U., arbitrary units. Molecular weight markers are depicted to the left. *p < 0.05 compared to control.

Figure 2. Activation of transcription factors linked to Bim regulation in the hippocampus after status epilepticus

(A) Representative Western blots (n = 2 per lane) showing expression of phosphorylated (p)FoxO1 declined following seizures. Blot below shows the non-phosphorylated form and Actin as a protein loading control. (B) Graph showing semi-quantitative analysis of pFoxO1 levels in the hippocampus. (C) Representative Western blots (n = 2 per lane) showing expression of CHOP and JNK1/2/3 isoforms. Note increased CHOP and pJNK levels. (D) Graph showing semi-quantitative analysis of CHOP levels in the hippocampus. (E) Graph showing semi-quantitative analysis of pJNK1/2/3. Data are from three independent experiments. *p < 0.05 compared to control (C, 4 h). (F) Representative Western blots (n = 1 per lane) showing hippocampal Bim expression at 24 h in control and KA-treated mice given either vehicle (V) or the JNK inhibitor SP600125 (SP). Note reduced Bim induction in seizure mice treated with SP600125. Actin is included as a protein loading control.

Figure 3. Bim expression declines in the less-damaged neocortex following seizures

(A) Photomicrographs (4× lens) show representative Fluoro-Jade B staining at 24 h in the neocortex of control mice and mice that underwent status epilepticus. Arrows denote cells (white) undergoing degeneration. Cortical layers are indicated. (B) Representative Western blot (n = 1 per lane) showing decreased expression of Bim after seizures. Actin is shown as a control for protein loading. Graph below shows semi-quantification of Bim protein levels in the neocortex. *p < 0.05 compared to control (C, 4 h). (C) Representative Western blots (n = 1 per lane) for pFoxO1, CHOP and actin and corresponding semi-quantification of protein levels for pFoxO1 in the neocortex. (D) Representative Western blots (n = 1 per lane) and graphs showing (top) pJNK1/2/3 and (bottom) total-JNK1/2/3 in the neocortex. All graph data are from three independent experiments. (E) Panels show representative 40× lens photomicrographs of Bim and TUNEL staining in control and neocortex 24 h after status epilepticus. Note decline in Bim staining and presence of scattered TUNEL-positive cells after seizures.

Figure 4. Genotype and phenotype analysis of bim^−/− mice

(A) Representative PCR showing genotyping of mice. (B) Representative Western blot (n = 2 per lane) confirming normal expression of KA receptor GluR6/7 in the mouse amygdala between wild-type (wt) and bim knockout mice. Actin is shown as a control for protein loading. (C) Representative Western blots of hippocampal samples confirming Bim deficiency in bim^−/− mice, while levels of a selection of other proteins are normal. (D) Representative photomicrographs of NeuN-stained CA3 subfields from wild-type and bim^−/− mice. (E) Graphs showing counts of NeuN-positive cells in various hippocampal fields. No differences were evident between mice of the two genotypes (n = 3 per group). (F) Graphs showing EEG data on seizure parameters between wild-type and bim^−/− mice following intra-amygdala KA microinjection. No differences were found between mice of the two genotypes (n = 9–10 per group).

Figure 5. Hippocampal but not neoortical seizure damage is reduced in bim^−/− mice

(A, B) Representative photomicrographs (4× lens) of Fluoro-Jade B staining within the ipsilateral hippocampus 72 h after status epilepticus in wild-type (wt) and bim^−/− mice. Note the reduction in numbers of damaged/dying cells within CA3 in bim^−/− mice compared to wild-type animals. (C–F) Representative photomicrographs (40× lens) of Fluoro-Jade B staining within the ipsilateral hippocampal CA3a and CA3b/c regions 72 h after status epilepticus in wild-type and bim^−/− mice. Note, the reduced numbers of Fluoro-Jade B positive cells in bim^−/− mice compared to wild-type animals. (G, H) Graphs quantifying numbers of Fluoro-Jade B stained cells in the hippocampal CA3 and the neocortex for each genotype of mice. Data are derived from n = 6–10 per group. *p < 0.05. ns, not significant. (I, J) Representative photomicrographs (4× lens) of Fluoro-Jade B staining within the ipsilateral neocortex 72 h after status epilepticus in wild-type and bim^−/− mice.

Figure 6. Bim depletion by short interfering RNA reduces KA-induced neuronal death in hippocampal organotypic cultures

(A) Representative photomicrographs (4× lens) show control and KA-treated cultures stained with propidium iodide (PI) at 24 h. Note, the significant cell death within hippocampal CA1 and CA3 (arrows). (B) Representative PI staining in KA-treated cultures at 24 h. Note, cell death is reduced in the cultures transfected with short interfering RNA targeting bim (bim siRNA) compared to cultures treated with a scrambled sequence (scram siRNA). (C) Representative PI staining in CA3 for each group. (D) Representative Western blots (n = 3 per lane) showing expression of Bim following KA treatment increases, and that bim siRNA reduces Bim protein levels. Blots below show levels of the KA receptor were not altered by the treatments, and probing for actin is provided as a control for equal protein loading. Data are derived from three independent experiments. (E, F) Graphs show quantification of cell death in CA1 and CA3 24 h after treatment. *p < 0.05 for comparison between cultures treated with KA plus bim siRNA compared to KA-treated cultures treated with scrambled siRNA.