Olianas Maria C, Dedoni Simona, Onali Pierluigi
Laboratory of Cellular and Molecular Pharmacology, Section of Neurosciences and Clinical Pharmacology, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy.
Br J Pharmacol. 2016 Oct;173(19):2910-28. doi: 10.1111/bph.13570. Epub 2016 Aug 26.
Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-β-induced neuronal apoptosis.
The effects of the ACh receptor agonist carbachol (CCh) on IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using western blots, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and overexpression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-β-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression.
In SH-SY5Y cells, CCh inhibited IFN-β-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 receptors, blocked by Gq/11 antagonist YM254890 and PKC inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by overexpression of ERK2 and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-β inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, Oxo-M reduced IFN-β-induced apoptosis; this effect was antagonized by blockade of M1 /M3 receptors and ERK1/2.
Stimulation of mAChRs counteracted IFN-β-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.
尽管I型干扰素（IFN）因其免疫调节、抗增殖和抗病毒特性在临床上具有重要作用，但它们也参与了多种神经退行性/神经炎症性疾病的发病机制。在本研究中，我们探讨了胆碱能刺激对IFN-β诱导的神经元凋亡的保护作用。
通过蛋白质免疫印迹法、免疫荧光法和细胞荧光测定法，研究了乙酰胆碱（ACh）受体激动剂卡巴胆碱（CCh）对IFN-β诱导的人神经母细胞瘤SH-SY5Y细胞凋亡的影响。使用选择性拮抗剂和小干扰RNA（siRNA）转染评估毒蕈碱型乙酰胆碱受体（mAChR）的参与情况。采用药理学抑制剂以及细胞外信号调节激酶2（ERK2）和ERK2组成型活性形式（ERK2-CA）的过表达来研究ERK1/2信号通路。通过检测裂解的半胱天冬酶3的表达，研究氧化震颤素-M（Oxo-M）对IFN-β诱导的小鼠海马神经元凋亡的影响。
在SH-SY5Y细胞中，CCh抑制IFN-β诱导的线粒体细胞色素c释放、半胱天冬酶9、7和3的激活、聚（ADP-核糖）聚合酶（PARP）裂解和DNA片段化。CCh的抗凋亡作用由M3受体介导，被Gq/11拮抗剂YM254890和蛋白激酶C（PKC）抑制剂Go 6983阻断，ERK1/2通路抑制可削弱其作用，ERK2过表达可增强其作用，ERK2-CA可模拟其作用。阻断c-Jun氨基末端激酶（JNK）激活可增强CCh的抗凋亡反应。IFN-β抑制JNK激活并上调CCh诱导的ERK1/2信号通路。在海马神经元中，Oxo-M减少IFN-β诱导的凋亡；M1/M3受体和ERK1/2阻断可拮抗这一作用。
刺激mAChR通过激活ERK1/2信号通路抵消IFN-β诱导的神经元凋亡。数据表明，激活与ERK1/2偶联的mAChR可能是预防IFN神经毒性的有效策略。
