Further evidence for the role of inositol trisphosphate as an excitotoxic death signal in hippocampal neurons

Introduction

We previously observed, under the video enhanced contrast-differential interference contrast (VEC-DIC) microscope, that the intact cultured neurons showed large nuclei containing an amorphous nucleoplasm, and that glutamate produced granulation inside the nucleus within 20 minutes ¹ . This nuclear change corresponds to DNA fragmentation ² . During this process, glutamate stimulates inositol trisphosphate (IP3) pathway, increases nucleoplasmic Ca2+ concentration ([Ca2+]n). N-methyl-D-aspartate receptor activation also stimulates IP3 pathway, and aggravates the process during glutamate excitotoxicity ² . On the other hand, mounting evidences revealed that mitochondrial dysfunction plays important roles during excitotoxic neuronal death. In this study, we examined whether mitochondrial toxin induces the nuclear change; and if so, we sought to determine the mechanism underlying the morphological changes.

Methods

Hippocampal neurons were obtained from one-day-old rats, and morphological changes were observed under a VEC-DIC microscope. Using a confocal laser microscope, mitochondrial membrane potential was measured with JC-1, a fluorescence indicator for mitochondrial membrane potential, changes in [Ca2+]n were assessed with fluo-3, a Ca2+-sensitive dye, and IP3 signal was detected with green fluorescent protein fused to the PH domain which translocates from the plasma membrane to the cytoplasm when IP3 increases.

Results

Within 20 min, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 1–10 μM), a potent mitochondrial uncoupler, dose-dependently decreased mitochondrial membrane potential and induced granulation inside the nucleus under the VEC-DIC microscope, which was identical to the findings induced by glutamate (100 μM–1 mM). Toxic dosage of FCCP increased [Ca2+]n before inducing the nuclear changes. A removal of Ca2+ from the medium did not affect the time course of both [Ca2+]n increase and the nuclear changes. Toxic dosage of FCCP increased intracellular IP3 concentration before inducing nuclear changes. When the neurons were permeabilized with β-escin (1 μM), an application of IP3 (110 μM) with EGTA (5 mM), a Ca2+ chelating agent, dose-dependently increased [Ca2+]n and induced rapid granular changes in the nucleus, which were inhibited by coadministration of heparin (5 U/ml), an IP3 receptor inhibitor, indicating that IP3 directly increased [Ca2+]n without extranuclear Ca2+.

Comments

Glutamate stimulates IP3 pathway, increases [Ca2+]n, and induces DNA fragmentation during the early phase of necrosis in the hippocampal neurons. Associated mitochondrial dysfunction also stimulates IP3 pathway, and aggravates the process of acute DNA fragmentation during glutamate excitotoxicity. Since the nucleus itself is a Ca2+ store and has IP3 receptors, IP3 can directly increase [Ca2+]n. This nuclear calcium signal controls a variety of nuclear functions, including gene transcription, DNA synthesis, and DNA repair. Probably, the increase in [Ca2+]n persisting above the physiological level results in the damage of nuclear DNA.