M-Calpain Activation Facilitates Seizure Induced KCC2 Down Regulation

Abstract

Potassium chloride co-transporter 2 (KCC2), a major chloride transporter that maintains GABA_A receptor inhibition in mature mammalian neurons, is down-regulated in the hippocampus during epileptogenesis. Impaired KCC2 function accelerates or facilitates seizure onset. Calpain, with two main subtypes of m- and μ-calpain, is a Ca²⁺-dependent cysteine protease that mediates the nonlysosomal degradation of KCC2. Although recent studies have demonstrated that calpain inhibitors exert antiepileptic and neuroprotective effects in animal models of acute and chronic epilepsy, whether calpain activation affects seizure induction through KCC2 degradation remains unknown. Our results showed that: (1) Blockade of calpain by non-selective calpain inhibitor MDL-28170 prevented convulsant stimulation induced KCC2 downregulation, and reduced the incidence and the severity of pentylenetetrazole (PTZ) induced seizures. (2) m-calpain, but not μ-calpain, inhibitor mimicked MDL-28170 effect on preventing KCC2 downregulation. (3) Phosphorylation of m-calpain has been significantly enhanced during seizure onset, which was partly mediated by the calcium independent MAPK/ERK signaling pathway activation. (4) MAPK/ERK signaling blockade also had similar effect as total calpain blockade on both KCC2 downregulation and animal seizure induction. The results indicate that upregulated m-calpain activation by MAPK/ERK during convulsant stimulation down regulates both cytoplasm- and membrane KCC2, and in turn facilitates seizure induction. This finding may provide a foundation for the development of highly effective antiepileptic drugs targeting of m-calpain.

Keywords: KCC2; MAPK/ERK; PTZ; calpain; epilepsy.

Figure 1

Electroencephalograph (EEG) and behavioral analysis showing improvements after MDL-28170 intervention on pentylenetetrazole (PTZ)-induced acute seizures. (A,B) typical EEG recordings from vehicle group (A) and MDL-28170 pre-treatment group (B) at selected time points. The lower traces are the enlarged traces from the dashed boxes of upper traces. The red dashed box marks an intermittent presence of high amplitude fast discharges (HAFDs) within the continuous rhythmic spike-wave activity. (C,D) EEG power from the recording of above traces was plotted against time, starting from the administration of PTZ and lasting for 30 min. The white arrow marks the HAFDs. (E,F) The static behavioral seizure scores over the 30 min period from two representative individual rats in the two groups illustrated in panel. (G) Proportion of rats with different seizure scores. R2, R3 and R5 represent the seizure score of Racine2, Racine3 and Racine5, respectively. (H) The average of the maximal behavior scores for rats from either PTZ or PTZ + MDL group. (I) Rate of animal with bursts and HAFDs within the two groups. *p < 0.05 compared with PTZ group; **p < 0.01 compared with PTZ group.

Figure 2

MDL-28170 reverses the down regulation of potassium chloride co-transporter 2 (KCC2) during seizure induction. (A) KCC2 immunostaining showing expression of KCC2 in membrane of hippocampal CA1 neurons after 0 Mg²⁺ (middle) or 0 Mg²⁺ + MDL-28170 (bottom) treatment, in comparison with vehicle control (left). Green and red signals represent the KCC2 and NeuN immunostaining, respectively. Scale bar: 20 μm. (B) The plasma membrane KCC2 (mKCC2) expression in hippocampus from PTZ alone or PTZ + MDL-21870 treated rats. The relative protein levels were normalized to the average of control group. (C) Spectrin breakdown product (mKCC2) expression from hippocampal slices. Slices were prepared from fresh brain and randomly divided into three different groups. KCC2 levels were normalized to control slices from the same preparation. (D) Spectrin breakdown product (SBDP)-145 level of 0 Mg²⁺ and 0 Mg²⁺ + MDL-28170 treated slices (total protein sample), normalized to control slices from the same preparation. *p < 0.05 compared with vehicle-PTZ group; **p < 0.01 compared with vehicle-PTZ group; ***p < 0.001 compared with vehicle-PTZ group; ^#p < 0.05 compared with 0 Mg²⁺ group; ^##p < 0.01 compared with 0 Mg²⁺ group.

Figure 3

Phosphor-serine activation of m-calpain dominants KCC2 regulation in 0 Mg²⁺ seizure model. (A,B) Immunoblotting detection of both plasma membrane (A) and cytoplasm (B) KCC2 expression level change in hippocampal slice 0 Mg²⁺ model treated with either μ-calpain or m-calpain selective inhibitor CI I and CI IV, respectively. (C) Expression of either μ- or m-calpain from seizure slices induced by 0 Mg²⁺. (D) Serine phosphorylation level by IP method. Naïve or 0 Mg²⁺ treated slices were used as inputs to form immune complex with μ- or m-calpain antibodies and detected by phosphor-serine antibody. Phosphor-serine levels were normalized to corresponded calpain levels. (E) Phosphor-serine level of m-calpain from 0 Mg²⁺, 0 Mg²⁺ + PD98059 or K252a treated slices. (F) Immunoblotting showed KCC2 level in 0 Mg²⁺ + MDL-28170 or PD98059 treated slices, compared with none treated or 0 Mg²⁺ treated alone slices. (G) KCC2 level of in vivo rats pre-treated with either MDL-28170 or SL-327 before PTZ kindling, in compared with vehicle PTZ treated rats. CI I, Calpain Inhibitor I; CI IV, Calpain Inhibitor IV; Calp, calpain; p-Ser, Phosphor-serine. *p < 0.05 compared with Ctrl group; **p < 0.01 compared with Ctrl group; ***p < 0.001 compared with Ctrl group; ^#p < 0.05 compared with 0 Mg²⁺ group; ^##p < 0.01 compared with either 0 Mg²⁺ or with PTZ group; ^###p < 0.001 compared with PTZ group.

Figure 4

The regulation of KCC2 by m-calpain is independent on [Ca²⁺]_i but is related to KCC2 endocytosis. (A) BAPTA is able to reverse 0 Mg²⁺ induced mKCC2 down-regulation, but failed to reverse BDNF induced mKCC2 down-regulation. (B) BAPTA partially reverses the 0 Mg²⁺ induced, but not BDNF induced Changes in cytoplasmic KCC2 (cKCC2) down-regulation. (C) Phosphor-serine level of m-calpain from 0 Mg²⁺, 0 Mg²⁺ + BAPTA, BDNF and BDNF + BAPTA treated slices. BAPTA intervention partially reverses the 0 Mg²⁺ induced, but not BDNF induced the increase of phosphor-serine level of m-calpain. (D–E) Tautomycetin (TMC) can reverse KCC2 down-regulation in plasm membrane (left), but the expression of KCC2 in cytoplasm remained significantly lower than DMSO group (right). In TMC + PD98059 co-treated group the expression of KCC2 is rescued to control level. *p < 0.05, **p < 0.01 compared with Ctrl group; ^#p < 0.05, ^##p < 0.01 compared with either 0 Mg²⁺ group.

Figure 5

SL-327 intervention improved the incoming seizure induced by PTZ in freely moving rats. (A) Typical EEG recording traces from SL-327 pre-treated rats. The lower traces are an enlarged drawing from the dashed boxes of upper traces. (B) EEG power from the recording of above trace was plotted against time, from PTZ administration to the following 30 min. The intensity of high frequency firing has been increasing in the first 10 min, but never reached to HAFDs during 30 min analysis in comparison with PTZ control rat described in Figure 1A. (C) Rate of animal with bursts and HAFDs from the two treating groups. (D,E) The static behavioral seizure scores over the 30 min period from the PTZ alone and PTZ + SL-327 groups illustrated in panel. (F) Proportion of rats with different seizure score. R3 and R5 represent the seizure score of Racine3 and Racine5, respectively. (G) The average of the maximal behavior scores for rats from either PTZ or SL-327 group. *p < 0.05 compared with PTZ group.

Figure 6

Drawing diagram showing the cellular mechanism of m-calpain phosphorylation involvement in seizure onset induced KCC2 down regulation. Both endocytosis and TrkB-MAPK/ERK–m-calpain pathways regulated KCC2 level in neurons.