Calcium-sensitive regulation of monoamine oxidase-A contributes to the production of peroxyradicals in hippocampal cultures: implications for Alzheimer disease-related pathology

Abstract

Background: Calcium (Ca2+) has recently been shown to selectively increase the activity of monoamine oxidase-A (MAO-A), a mitochondria-bound enzyme that generates peroxyradicals as a natural by-product of the deamination of neurotransmitters such as serotonin. It has also been suggested that increased intracellular free Ca2+ levels as well as MAO-A may be contributing to the oxidative stress associated with Alzheimer disease (AD).

Results: Incubation with Ca2+ selectively increases MAO-A enzymatic activity in protein extracts from mouse hippocampal HT-22 cell cultures. Treatment of HT-22 cultures with the Ca2+ ionophore A23187 also increases MAO-A activity, whereas overexpression of calbindin-D28K (CB-28K), a Ca2+-binding protein in brain that is greatly reduced in AD, decreases MAO-A activity. The effects of A23187 and CB-28K are both independent of any change in MAO-A protein or gene expression. The toxicity (via production of peroxyradicals and/or chromatin condensation) associated with either A23187 or the AD-related beta-amyloid peptide, which also increases free intracellular Ca2+, is attenuated by MAO-A inhibition in HT-22 cells as well as in primary hippocampal cultures.

Conclusion: These data suggest that increases in intracellular Ca2+ availability could contribute to a MAO-A-mediated mechanism with a role in AD-related oxidative stress.

Figure 1

Effect of Ca²⁺ on MAO activity in the mouse hippocampal HT-22 cell line. HT-22 cell lysates (100 μg/100 μL; n = 4) were incubated with increasing concentrations of Ca²⁺ and assayed radioenzymatically for (A) MAO-A and (B) MAO-B activities. (C) HT-22 lysates (n = 3) were incubated with either Ca²⁺ or the Ca²⁺ antagonist Mg²⁺ (1 mM) or co-incubated with Ca²⁺ (1 mM) and increasing concentrations of Mg²⁺ to test for the potential of Mg²⁺ to block Ca²⁺ (1 mM)-sensitive MAO-A activity. **: P < 0.01, ***: P < 0.001 vs. control. Data represent mean ± SD.

Figure 2

Effect of Ca²⁺ on MAO-A substrate kinetics in HT-22 hippocampal cell line. (A) HT-22 cell lysates (100 μg/100 μL; n = 3) were incubated with increasing concentrations of substrate, e.g. [¹⁴C]-5-HT in the absence (○) or presence (●) of Ca²⁺ (1 mM). The individual curves represent data pooled from three independent experiments. Data are presented as mean ± SD. (B) The highlighted part of the curves in (A) is expanded to demonstrate the change in K_m. (C) Data presented on a double-reciprocal plot clearly demonstrate a change in slope (e.g. K_m/V_max) with no change in y-intercept (e.g. 1/V_max).

Figure 3

The Ca²⁺ ionophore A23187 increases MAO-A activity. (A) Levels of free intracellular Ca²⁺, [Ca²⁺]_i, in HT-22 cells treated with A23187 (5 μM: 30 min) were determined using the Ca²⁺-binding Fluo-3 AM fluorescent dye. (B) MAO-A and MAO-B activities were assessed radioenzymatically in A23187-treated cell cultures (*P < 0.05 versus control levels). (C) MAO-A protein expression was determined in SDS-PAGE resolved total cell lysates. Levels of β-actin demonstrate equal protein loading. (D) Densitometry graph representing mao-A gene expression (as a ratio to GAPDH expression) determined using semi-quantitative RT-PCR amplification of mRNA extracted from A23187-treated cell cultures. (E) Representative RT-PCR amplification fragments. (F) In similarly-treated cells, the production of ROS was assessed using the H₂O₂-binding DCF fluorogen. A parallel series of cell cultures were pre-treated with the specific MAO-A inhibitor, clorgyline (CLG; 100 μM, 1 h). Data represent mean ± SD.

Figure 4

Overexpression of the Ca²⁺-binding protein CB-28K decreases MAO-A activity. HT-22 cells were transfected with the pREP plasmid (Vector) or the pREP-CB-28K expression vector (CB-28K). (A) The effect of CB-28K overexpression (24 h) on free intracellular Ca²⁺, [Ca²⁺]_i, and on ROS production was examined. (note, the relative fluorescence intensity used for this set of ROS experiments was increased intentionally so as to avoid a "floor" effect, i.e. so that a decrease in ROS production could be demonstrated in CB-28K-overexpressing cells). (B) MAO-A and MAO-B activities were assessed radioenzymatically in corresponding cell lysates (*P < 0.05 versus control levels). (C) SDS-PAGE-resolved total cellular proteins were probed for CB-28K overexpression and for the expression of MAO-A and β-actin (used as a loading control). (D) Densitometry graph representing mao-A gene expression (relative to β-actin expression) determined by semi-quantitative RT-PCR, represented in (E). Data are presented as mean ± SD.

Figure 5

ROS production induced by the Alzheimer disease-related peptide, Aβ, is diminished by MAO-A inhibition. HT-22 cells were treated with Aβ(1–40) (30 μM, 48 h). The effect of Aβ(1–40) on (A) free intracellular Ca²⁺, assessed using FLUO-3AM fluorescence, and on the production of ROS, assessed using the H₂O₂-binding DCF fluorogen. (B) Hoechst staining of primary hippocampal cell cultures was used to determine the proportion of cells exhibiting chromatin condensation (an indication of apoptotic cell death: arrows) in Aβ(1–42)-treated cultures. In both HT-22 cultures (A) and primary neuronal cultures (B), the effect of Aβ was reversed by pretreatment with the specific MAO-A inhibitor, clorgyline (CLG; 100 μM, 1 h). (C) Number of primary hippocampal cells (expressed as a percentage of control) that exhibit chromatin condensation following treatment with Aβ and/or CLG (groups shown in B). ***: P < 0.001 vs. control. ##: P < 0.01 between the indicated groups. Data are presented as mean ± SD.