Transforming growth factor- α's effects on astroglial–cholinergic cell interactions in the medial septal area in vitro are mediated by α2-macroglobulin

Abstract

We reported previously that two epidermal growth factor receptor ligands, epidermal growth factor and transforming growth factor- α, inhibit medial septal cholinergic cell phenotypic expression (choline acetyltransferase and acetylcholinesterase activities) in vitro indirectly via (a) soluble molecule(s) released from astrocytes [Kenigsberg R. L. et al. (1992) Neuroscience 50, 85–97; Kenigsberg R. L. and Mazzoni I. E. (1995) J. Neurosci. Res. 41, 734–744; Mazzoni I. E. and Kenigsberg R. L. (1996) Brain Res. 707, 88–99]. In the present study, we found that this response to transforming growth factor- α is mediated, for the most part, by α 2-macroglobulin, a potent protease inhibitor with a wide spectrum of biological activities. In this regard, the effects of transforming growth factor- α on cholinergic cells can be blocked with immunoneutralizing antibodies raised against α 2-macroglobulin. Furthermore, western blot analysis reveals that although α 2-macroglobulin is present in conditioned media from control septal cultures, it is more abundant in those treated with transforming growth factor- α. In addition, exogenous α 2-macroglobulin, both in its native and trypsin-activated forms, can mimic transforming growth factor- α's effects on septal cholinergic cell expression. However, while the native antiprotease can slightly but significantly decrease choline acetyltransferase activity, trypsin-activated α 2-macroglobulin, in the nanomolar range, induces as marked a decrease in this enzyme activity as that noted with transforming growth factor- α. Furthermore, trypsin-activated α 2-macroglobulin, like epidermal growth factor/transforming growth factor- α, decreases choline acetyltransferase activity by arresting its spontaneous increase that occurs with time in culture, does so in a reversible manner and is not neurotoxic. In addition, trypsin-activated α 2-macroglobulin, in the nanomolar range, can affect choline acetyltransferase in a dual manner, up-regulating it at low concentrations while down-regulating it at higher ones. These responses are identical in mixed neuronal–glial and pure neuronal septal cultures. Furthermore, when concentrations of trypsin-activated α 2-macroglobulin, which alone decrease choline acetyltransferase, are added simultaneously with nerve growth factor, they serve to potentiate the nerve growth factor-induced increase in enzymatic activity. As GABAergic cell expression is not affected by α 2-macroglobulin, it appears that the effects of this protease inhibitor on medial septal neuronal expression are neurotransmitter-specific. Finally, trypsin-activated but not native α 2-macroglobulin promotes a dose-dependent aggregation of the septal neurons. This change in morphology, however, is not related to those noted in choline acetyltransferase activity. In summary, these data suggest that the expression of α 2-macroglobulin in astroglia from the medial septal nucleus can be controlled by epidermal growth factor receptor ligands to impact the functioning of basal forebrain cholinergic neurons.