Proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy: potential targets in calcium regulatory network

Abstract

Herein proteomic profiling of the rat hippocampus from the kindling and pilocarpine models of epilepsy was performed to achieve new potential targets for treating epileptic seizures. A total of 144 differently expressed proteins in both left and right hippocampi by two-dimensional electrophoresis coupled to matrix-assisted laser desorption-mass spectrometry were identified across the rat models of epilepsy. Based on network analysis, the majority of differentially expressed proteins were associated with Ca²⁺ homeostasis. Changes in ADP-ribosyl cyclase (ADPRC), lysophosphatidic acid receptor 3 (LPAR3), calreticulin, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), synaptosomal nerve-associated protein 25 (SNAP 25) and transgelin 3 proteins were probed by Western blot analysis and validated using immunohistochemistry. Inhibition of calcium influx by 8-Bromo-cADP-Ribose (8-Br-cADPR) and 2-Aminoethyl diphenylborinate (2-APB) which act via the ADPRC and LPAR3, respectively, attenuated epileptic seizures. Considering a wide range of molecular events and effective role of calcium homeostasis in epilepsy, polypharmacy with multiple realistic targets should be further explored to reach the most effective treatments.

Figure 1

2-DE analysis of the hippocampus proteome. The timeline of both control and epileptic tissue sampling is shown in each panel. One milligram of total proteins of each sample was separated by 2-DE on a pH 3–10 linear IPG strip in the first dimension and on a 12.5% SDS–PAGE gel in the second dimension. Representative gels from control tissues were showed in (A): the left (A1) and the right hippocampus (A2); panel (B) is related to pilocarpine model: the left (B1) and the right hippocampus (B2); panel (C) showed gels related to PTZ kindling model: The left (C1) and the right hippocampus (C2) and panel (D) showed proteome profile of left (D1) and right (D2) hippocampus related to electrical kindling model. Differently expressed spots were analyzed by ImageMaster 2D Platinum v7.0 and showed by arrows.

Figure 2

The process of detection and recognition of different spots. Differently expressed spots in each epilepsy model rather than control were visualized and recognized by MALDI–TOF–TOF/MS spectrometer method after trypsin digestion. Commonalities and differences of changed proteins categorized between three models as a Venn diagram. The short name of each identified protein for each group is indicated.

Figure 3

Molecular interaction network with 139 nodes consists of 125 proteins and 14 functions. The interaction numbers are 196 which are classified as two types: inhibiting (blue) and activating (red). Network consists of 95 detected proteins and some bridge nodes. Expressed proteins are clarified in 4 of 14 functions: Ca²⁺ influx, apoptosis, inflammation and membrane depolarization.

Figure 4

Immunoblotting analysis. (A) 2-DE gel images of six protein spots of interest denoted by an arrow in each panel. Each panel shows an expanded 2-D gel view and ranked from the left to the right: the left (CL) and the right hippocampus (CR) of the control group; the left (PTZL) and the right hippocampus (PTZR) of the PTZ group; the left (ElecL) and the right hippocampus (ElecR) of the electrical kindling group; and the left (PiL) and the right hippocampus (PiR) of the pilocarpine group. (B) Western blot analysis validates the differential expression of ADPRC1, Calreticulin, SNAP 25, UCH-L1, LPAR3 and transgelin 3 in the control and the epileptic groups. Beta-actin was used for normalization. (C) The intensity of bands was quantified by ImagJ software. The data were expressed as mean ± S.E.M. The letters above the columns (a-h) indicate significant differences (P < 0.05) using the Duncan’s multiple range test so, data that showed with the same letter don’t have significant differences. (D) Representative photographs of immune-histochemical analysis of the expression levels of the ADPRC1, the calreticulin, the SNAP 25 and the UCH-L1 in control and epileptic groups.

Figure 5

Blocking of calcium influx inhibits seizure in both in vivo and in vitro seizure models. (A) Suppression of the high-K⁺/low-Mg²⁺ induced epileptiform activity in the CA1 region of the hippocampal slice in the presence of 8-Br-cADPR and 2-APB. Inhibition of epileptiform discharges by reducing of the spiking numbers (B), spiking duration (C), onset latency of spiking (D) and spiking rate (E), in the presence of the both Ca²⁺ channels inhibitors, particularly in the presence of 8-Br-cADPR. 8-Br-cADPR suppresses epileptic seizures in the PTZ-kindled rats (F) as indicated by a significant decrease in seizure duration (G) and an insignificant effect on seizure latency (H). Data were expressed as mean ± S.E.M. Differences between groups were analyzed by one‑way analysis of variance (ANOVA). *Represents a significant difference as compared to control group (P < 0.05).

Figure 6

Schematic overview of intracellular Ca²⁺ cycling. (A) The most prominent homeostatic event in epileptic hippocampus is the accumulation of intracellular calcium. (B) General scheme of Ca²⁺ influx pathways indicating the disrupted pathways in epileptic hippocampus: 1. NMDA glutamate receptor 2. AMPA glutamate receptor 3. Calcium influx activated by ADPRC as a second messenger, and 4. Ca²⁺-release channels from endoplasmic reticulum including LPAR3 receptor pathway.