Endothelin-1 is over-expressed in amyotrophic lateral sclerosis and induces motor neuron cell death.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of motor 29 neurons (MNs) and astrogliosis. Recent evidences suggest that factors secreted by activated astrocytes might 30 contribute to degeneration of MNs. We focused on endothelin-1 (ET-1), a peptide which is strongly up- 31 regulated in reactive astrocytes under different pathological conditions. We show that ET-1 is abundantly 32 expressed by reactive astrocytes in the spinal cord of the G93A mouse model and sporadic ALS patients. To test 33 if ET-1 might play a role in degeneration of MNs, we investigated its effect on MN survival in an in vitro model 34 of mixed rat spinal cord cultures (MSCs) enriched of astrocytes exhibiting a reactive phenotype. ET-1 exerted a 35 toxic effect on MNs in a time- and concentration-dependent manner, with an exposure to 100-200 nM ET-1 36 for 48 h resulting in 40-50% MN cell death. Importantly, ET-1 did not induce MN degeneration when adminis- 37 tered on cultures treated with AraC (5 ?M) or grown in a serum-freemediumthat did not favor astrocyte prolif- 38 eration and reactivity.We found that both ETA and ETB receptors are enriched in astrocytes in MSCs. The ET-1 39 toxic effect was mimicked by ET-3 (100 nM) and Sarafotoxin S6C (10 nM), two selective agonists of 40 endothelin-B receptors, andwas not additivewith that of ET-3 suggesting the involvement of ETB receptors. Sur- 41 prisingly, however, the ET-1 effect persisted in the presence of the ETB receptor antagonist BQ788 (200 nM- 42 2 ?M) andwas slightly reversed by the ETA receptor antagonist BQ123 (2 ?M), suggesting an atypical pharmaco- 43 logical profile of the astrocytic receptors responsible for ET-1 toxicity. The ET-1 effect was not undone by the 44 ionotropic glutamate receptor AMPA antagonist GYKI 52466 (20 ?M), indicating that it is not caused by an in- 45 creased glutamate release. Conversely, a 48-hour ET-1 treatment increased MN cell death induced by acute ex- 46 posure to AMPA (50 ?M), which is indicative of two distinct pathways leading to neuronal death. 47 Altogether these results indicate that ET-1 exerts a toxic effect on culturedMNs through mechanisms mediated 48 by reactive astrocytes and suggest that ET-1may contribute toMN degeneration in ALS. Thus, a treatment aimed 49 at lowering ET-1 levels or antagonizing its effect might be envisaged as a potential therapeutic strategy to slow 50 down MN degeneration in this devastating disease.