Acidosis-induced zinc-dependent death of cultured cerebellar granule neurons

Abstract

Severe acidosis caused death of cultured cerebellar granule neurons (CGNs). Acidosis was accompanied by a progressive increase of the intracellular zinc ions ([Zn(2+)](i)) and decrease of [Ca(2+)](i). Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), prevented the increase of [Zn(2+)](i) and acidosis-induced neuronal death. However, neuronal death was insensitive to blockade of ASIC1 channels with amiloride, as CGNs display considerably lower expression of ASIC1a than other neurons. The antioxidant trolox and menadione significantly protected neurons from acidotic death. Earlier, we demonstrated that menadione rescues neurons from the deleterious effect of inhibition of mitochondrial complex I (Isaev et al. Neuroreport 15:2227-2231, 2004). We speculate that excessive Zn(2+)-dependent production of reactive oxygen species by mitochondrial complex I may be a general motive for the induction of cell death in CGNs under acidotic conditions.

Fig. 1

Acidosis (24 h) induces death of the cultivated cerebellar granule neurons (black bars), with amiloride being ineffective (white bars). * P < 0.01, vs. pH 7.3

Fig. 2

Quantitative real-time polymerase chain reaction analysis of ASIC1a mRNA levels in primary cerebellar granule neurons (CGN) compared to primary brain cortical neurons (CTX). ASIC1a expression in CGN (11.6%) was normalized to expression in CTX (100%)

Fig. 3

Acidosis (30 min) leads to (a) the decrease of fluorescence of the Ca²⁺-sensitive probe Fluo-4 and (b) the increase of fluorescence of the Zn²⁺-sensitive probe FluoZin-3 in the cerebellar granule neurons * P < 0.05, vs. pH 7.3

Fig. 4

Acidosis (30 min) leads to the rise of fluorescence of the Zn²⁺-sensitive probe (FluoZin-3), which is abolished by a specific zinc chelator TPEN. a pH 7.3, b pH 6.0, c pH 7.3 + TPEN, d pH 6.0 + TPEN. a–d, FluoZin-3 fluorescence, a1–d1; phase contrast. Bar 10 μm

Fig. 5

Zinc chelator TPEN (a) and antioxidant trolox (b) increase the cerebellar granule neurons survival under pH 6.0, 24 h. * P < 0.05 when compared with pH 7.3; ** P < 0.001 when compared white and black bars under corresponding pH value

Fig. 6

Menadione increases cerebellar granule neurons survival under the toxic effect (24 h) of acidosis (a) and effect of extracellular Zn²⁺ addition (b).* P < 0.01 when compared with pH 7.3 (a) or when compared with zinc concentration “0” (b); **P < 0.001 when compared white and black bars under corresponding pH value or zinc concentration