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

The role of ß-amyloid (Aß) in the pathogenesis of Alzheimer's disease (AD) is still considered crucial. The state of Aß aggregation is critical in promoting neuronal loss and neuronal function impairment. Recently, we demonstrated that Acetylcholine (ACh) is neuroprotective against the toxic effects of Aß in the cholinergic LAN-2 cells. In biophysical experiments, ACh promotes the soluble Aß peptide conformation rather than the aggregation-prone ß-sheet conformation. In order to better understand the biological role of ACh in AD, we studied the effect of Aß 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 Aß in both undifferentiated and RA-differentiated TB cells. Our results show that TB cells are responsive to Aß. Moreover, in undifferentiated cells 1 h treatment with Aß 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 Aß-treated and non-treated cells after 4 and 7 days in vitro. The RA-differentiated cells are not sensitive to Aß. In TB cell line ACh is able to blunt the effects of Aß. The ability of ACh to protect non-cholinergic cells against Aß reinforces the hypothesis that, in addition to its role in cholinergic transmission, ACh could also act as a neuroprotective agent.

Treating TB human neuroectodermal cell line with retinoic acid induces the appearance of neuron-like voltage-gated ionic currents.

TB is a cell line derived from the cerebrospinal fluid sample of a patient with primary leptomeningeal melanomatosis. Our previous immunological and ultrastructural analysis revealed that TB cells differentiate towards a neuronal phenotype when grown in vitro up to 7 days in presence of 10 µM all-trans retinoic acid (RA). Recently, we reported that TB cells are sensitive to the cytotoxic effects of ß-amyloid peptides, activating the cytosolic phospholipase A2. To date, it is not known if RA, in addition to inducing morphological changes, also causes functional modification in TB cells, by regulating voltage-gated ionic currents. To this purpose, we performed electrophysiological characterization of undifferentiated (TB) and differentiated (RA-TB) cells by means of whole-cell patch clamp recordings. Upon depolarizing stimuli, both groups displayed voltage-gated K+ outward currents of similar amplitude. By contrast, the low amplitude voltage-gated Na+ currents recorded in undifferentiated TB cells were largely up-regulated by RA exposure. This current was strongly reduced by TTX and lidocaine and completely abolished by removal of extracellular sodium. Furthermore, treatment with RA caused the appearance of a late-onset inward current carried by Ca2+ ions in a subpopulation of TB cells. This current was not affected by removal of extracellular Na+ and was completely blocked by Cd2+, a broad-spectrum blocker of Ca2+ currents. Altogether, our results indicate that RA-differentiation of TB cells induces functional changes by augmenting the amplitude of voltage-gated sodium current and by inducing, in a subpopulation of treated cells, the appearance of a voltage-gated calcium current.