Institute of Neurological Sciences, National Research Council (ISN-CNR), Catania, Italy; PhD Program in Neurobiology, University of Catania, Catania, Italy.
Institute of Neurological Sciences, National Research Council (ISN-CNR), Catania, Italy.
Neurobiol Dis. 2014 May;65:160-71. doi: 10.1016/j.nbd.2014.01.002. Epub 2014 Jan 11.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of motor neurons (MNs) and astrogliosis. Recent evidence suggests that factors secreted by activated astrocytes might contribute to degeneration of MNs. We focused on endothelin-1 (ET-1), a peptide which is strongly up-regulated in reactive astrocytes under different pathological conditions. We show that ET-1 is abundantly expressed by reactive astrocytes in the spinal cord of the SOD1-G93A mouse model and sporadic ALS patients. To test if ET-1 might play a role in degeneration of MNs, we investigated its effect on MN survival in an in vitro model of mixed rat spinal cord cultures (MSCs) enriched of astrocytes exhibiting a reactive phenotype. ET-1 exerted a toxic effect on MNs in a time- and concentration-dependent manner, with an exposure to 100-200nM ET-1 for 48h resulting in 40-50% MN cell death. Importantly, ET-1 did not induce MN degeneration when administered on cultures treated with AraC (5μM) or grown in a serum-free medium that did not favor astrocyte proliferation and reactivity. We found that both ETA and ETB receptors are enriched in astrocytes in MSCs. The ET-1 toxic effect was mimicked by ET-3 (100nM) and sarafotoxin S6c (10nM), two selective agonists of endothelin-B receptors, and was not additive with that of ET-3 suggesting the involvement of ETB receptors. Surprisingly, however, the ET-1 effect persisted in the presence of the ETB receptor antagonist BQ-788 (200nM-2μM) and was slightly reversed by the ETA receptor antagonist BQ-123 (2μM), suggesting an atypical pharmacological profile of the astrocytic receptors responsible for ET-1 toxicity. The ET-1 effect was not undone by the ionotropic glutamate receptor AMPA antagonist GYKI 52466 (20μM), indicating that it is not caused by an increased glutamate release. Conversely, a 48-hour ET-1 treatment increased MN cell death induced by acute exposure to AMPA (50μM), which is indicative of two distinct pathways leading to neuronal death. Altogether these results indicate that ET-1 exerts a toxic effect on cultured MNs through mechanisms mediated by reactive astrocytes and suggest that ET-1 may contribute to MN degeneration in ALS. Thus, a treatment aimed at lowering ET-1 levels or antagonizing its effect might be envisaged as a potential therapeutic strategy to slow down MN degeneration in this devastating disease.
肌萎缩侧索硬化症（ALS）是一种神经退行性疾病，其特征是运动神经元（MNs）的进行性丧失和星形胶质细胞增生。最近的证据表明，激活的星形胶质细胞分泌的因子可能有助于 MNs 的退化。我们专注于内皮素-1（ET-1），一种在不同病理条件下强烈上调的肽。我们发现 ET-1 在 SOD1-G93A 小鼠模型和散发性 ALS 患者的脊髓中大量表达于反应性星形胶质细胞。为了测试 ET-1 是否可能在 MNs 的退化中发挥作用，我们在富含表现出反应性表型的星形胶质细胞的大鼠脊髓混合培养物（MSCs）的体外模型中研究了其对 MN 存活的影响。ET-1 以时间和浓度依赖的方式对 MNs 产生毒性作用，用 100-200nM ET-1 处理 48 小时会导致 40-50%的 MN 细胞死亡。重要的是，当用 AraC（5μM）处理或在不有利于星形胶质细胞增殖和反应性的无血清培养基中生长的培养物中给予 ET-1 时，它不会诱导 MN 退化。我们发现 ET-1 毒性作用的 ETB 受体在 MSCs 中的星形胶质细胞中富集。内皮素-B 受体的两种选择性激动剂 ET-3（100nM）和 Sarafotoxin S6c（10nM）模拟了 ET-1 的毒性作用，并且与 ET-3 的毒性作用没有相加作用，表明 ETB 受体的参与。然而，令人惊讶的是，在存在 ETB 受体拮抗剂 BQ-788（200nM-2μM）的情况下，ET-1 的作用仍然持续存在，并且 ETA 受体拮抗剂 BQ-123（2μM）略微逆转了 ET-1 的作用，表明负责 ET-1 毒性的星形胶质细胞受体具有非典型的药理学特征。ET-1 作用不能被离子型谷氨酸受体 AMPA 拮抗剂 GYKI 52466（20μM）消除，表明它不是由谷氨酸释放增加引起的。相反，48 小时 ET-1 处理增加了急性暴露于 AMPA（50μM）诱导的 MN 细胞死亡，这表明有两条不同的途径导致神经元死亡。总之，这些结果表明 ET-1 通过反应性星形胶质细胞介导的机制对培养的 MNs 产生毒性作用，并表明 ET-1 可能有助于 ALS 中的 MN 退化。因此，降低 ET-1 水平或拮抗其作用的治疗方法可能被视为减缓这种毁灭性疾病中 MN 退化的潜在治疗策略。
