Endothelin-1 induces degeneration of cultured motor neurons: underlying mechanisms

AIMS: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons (MNs) and astrogliosis. Factors secreted by activated astrocytes might contribute to MN degeneration. We focused on endothelin-1 (ET-1), a peptide strongly up-regulated in reactive astrocytes under pathological conditions. We have previously found that ET-1 i) is abundantly expressed by reactive astrocytes in the spinal cord of SOD-1 G93A mice and ALS patients and ii) exerts a toxic effect on cultured MNs through activation of astrocytic ET receptors (Ranno et al., 2014). The present study is aimed at identifying the mechanisms underlying ET-1 toxicity. We focused on cell survival pathways, such as phosphatidylinositide 3-kinase (PI3K) pathway and/or inflammatory processes. METHODS: Mixed spinal cord cultures were treated with ET-1 (100 nM for 48 h) with/without LPS (40 ng/ml) or inhibitors of PI3K (LY294002 50 µM) and nitric oxide (NO) synthase (L-NAME 40µM). We performed immunocytochemistry using SMI-32 antibody(Sternberger Monoclonal) to label MNs and counted the number of surviving MNs after treatments. RESULTS: Inhibition of PI3K pathway caused MN death, as expected; however, exposure to ET-1 in the presence of LY294002 did not result in a further increase of MN death. Similarly, LPS induced MN death but its effect was not additive with that of ET-1. Furthermore, the effect of ET-1 was not reversed by L-NAME. CONCLUSIONS: ET-1 may cause neuronal death by a mechanism that involves the PI3K pathway. An inflammatory component may account for the ET-1 toxicity, but does not involve NO production.