Effects of Acetylcholine on β-Amyloid-Induced cPLA2 Activation in the TB Neuroectodermal Cell Line: Implications for the Pathogenesis of Alzheimer's Disease

Abstract

The role of β-amyloid (Aβ) in the pathogenesis of Alzheimer's disease (AD) is still considered crucial. The state of Aβ aggregation is critical in promoting neuronal loss and neuronal function impairment. Recently, we demonstrated that Acetylcholine (ACh) is neuroprotective against the toxic effects of Aβ in the cholinergic LAN-2 cells. In biophysical experiments, ACh promotes the soluble Aβ peptide conformation rather than the aggregation-prone β-sheet conformation. In order to better understand the biological role of ACh in AD, we studied the effect of Aβ on the phosphorylation of the cytosolic phospholipase A2 (cPLA2) in the TB neuroectodermal cell line, which differentiates toward a neuronal phenotype when cultured in the presence of retinoic acid (RA). We chose the phosphorylated form of cPLA2 (Ser505, Phospho-cPLA2) as a biomarker to test the influence of ACh on the effects of Aβ in both undifferentiated and RA-differentiated TB cells. Our results show that TB cells are responsive to Aβ. Moreover, in undifferentiated cells 1 h treatment with Aβ induces a 2.5-fold increase of the Phospho-cPLA2 level compared to the control after 24 h in vitro, while no significant difference is observed between Aβ-treated and non-treated cells after 4 and 7 days in vitro. The RA-differentiated cells are not sensitive to Aβ. In TB cell line ACh is able to blunt the effects of Aβ. The ability of ACh to protect non-cholinergic cells against Aβ reinforces the hypothesis that, in addition to its role in cholinergic transmission, ACh could also act as a neuroprotective agent.

Keywords: Acetylcholine; Alzheimer’s disease; Differentiation; Phospholipase A2; TB cell line; β-Amyloid.

Fig. 1

Influence of differentiation on the level of phospho-cPLA2. Total proteins extracted from each sample were loaded (20 µg) on a pre-formed gel in a polyacrylamide gradient under denaturing conditions (SDS-PAGE). The Western blot analysis using a primary antibody specific for the phosphorylated form of cPLA2 (Ser505, Phospho-cPLA2) showed no significant differences between undifferentiated (−RA) and 10 µM RA-differentiated (+RA) TB cells up to 7 days in culture. The housekeeping ribosomal protein RPL7 was used as loading control. Details in the text

Fig. 2

Sensitivity of TB cells to Aβ. Western blot analysis on the total proteins extracted from undifferentiated TB cells treated for 1 h with 5 µM and 20 µM Aβ_25–35 after 24 h in culture. 5 µM Aβ failed to produce any significant effect on the level of the phosphorylated form of cPLA2, while 20 µM Aβ increased the level of the enzyme roughly 1.5 fold compared to the control (Ctrl). The housekeeping ribosomal protein RPL7 was used as loading control. Details in the text

Fig. 3

Influence of differentiation with retinoic acid on Aβ-induced cPLA2 activation. Western blot analysis on total proteins extracted from undifferentiated (−RA) and 10 µM RA-differentiated (+RA) TB cells grown up to 7 days in vitro and treated for 1 h with 20 µM Aβ_25–35 (+Aβ) after 1, 4, and 7 days in culture. In undifferentiated cells, Aβ increased the phosphorylation of cPLA2 2.5 fold compared to the control (−Aβ) after 24 h in vitro, while after 4 and 7 days in vitro no significant difference was observed between Aβ-treated and non-treated cells. In RA-differentiated cells, Aβ did not affect the level of cPLA2 phosphorylation at any of the three tested time points. The housekeeping ribosomal protein RPL7 was used as loading control. Details in the text

Fig. 4

Effects of ACh on Aβ-induced cPLA2 activation. Western blot analysis on total proteins extracted from TB cells treated for 1 h with 20 µM Aβ_25–35 and 25 µM ACh after 24 h in vitro without RA. Aβ (+Aβ −ACh) increased the phosphorylation of cPLA2 by about 60% compared to the negative control (−Aβ −ACh), while 25 µM ACh blunted this cytotoxic effect (+Aβ +ACh). ACh alone (−Aβ +ACh) had no effects on cPLA2 activation. The housekeeping ribosomal protein RPL7 was used as loading control. Details in the text

Fig. 5

Effects of ACh on Aβ-induced cPLA2 activation. Western blot analysis on total proteins extracted from TB cells treated for 1 h with 20 µM Aβ_25–35 and 25 µM ACh after 24 h in vitro in presence of RA. In this conditions Aβ (+Aβ −ACh) did not affect the level of cPLA2 phosphorylation compared to the negative control (−Aβ −ACh). ACh alone (−Aβ +ACh) had no effects on TB cells. The housekeeping ribosomal protein RPL7 was used as loading control. Details in the text