Drannik Anna, Martin Joan, Peterson Randy, Ma Xiaoxing, Jiang Fan, Turnbull John
Department of Medicine, Division of Neurology, McMaster University, Hamilton, Ontario, Canada.
Life Sciences Services, SGS Life Sciences Services Canada Mississauga, Ontario, Canada.
PLoS One. 2017 Feb 7;12(2):e0171668. doi: 10.1371/journal.pone.0171668. eCollection 2017.
Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to play an important role in adult life by contributing to cell proliferation and differentiation, maintaining blood-brain barrier integrity, and being cytoprotective against oxidative and excitotoxic stress, all features of importance in amyotrophic lateral sclerosis (ALS). ALS is a fatal disease characterized by selective loss of motor neurons due to poorly understood mechanisms. Evidence indicates that Shh might play an important role in ALS, and that Shh signaling might be also adversely affected in ALS. Since little is known about the functional status of Shh pathway in patients with ALS, we therefore sought to determine whether Shh protein levels or biological activity in cerebrospinal fluid (CSF) was less in ALS patients than controls, and whether these measures could be correlated with ALS disease severity and disease progression, and with other CSF analytes of biological interest in ALS. Comparing Shh levels in the CSF of normal controls (n = 13), neurological controls (n = 12), and ALS patients (n = 9) measured by ELISA, we found that CSF Shh levels were not different between controls and ALS patients. However, when assessing Shh biological activity in CSF using in vitro cell-based assays, which measure Shh activity as inducible Gli-driven luminescence, we found that in the presence of exogenous recombinant Shh or the Shh agonist, purmorphamine, the inducible activity of CSF was significantly augmented in the control groups as expected, but not in the ALS group, suggesting the presence of an inhibitor of Shh signaling in ALS CSF samples. Since purmorphamine acts on Smoothened, downstream of Shh and its receptor Patched, the inhibitory action is downstream of Smoothened. Our results also demonstrated that while the inhibitory effect of ALS CSF on Shh signaling did not correlate significantly with ALS disease characteristics, the levels of IL-1β and TNF-α did. In addition to being significantly elevated in ALS CSF, these cytokines negatively correlated with the disease duration, whereas GDF11 was a favorable predictor of ALS clinical score. We also found that TNF-α significantly inhibited Shh biological activity in vitro, potentially suggesting a novel role of TNF-α in ALS pathogenesis. Collectively, this is the first report demonstrating that Shh signaling in CSF of ALS patients is compromised.
音猬因子（Shh）是一种对神经系统发育至关重要的形态发生素，在成年期通过促进细胞增殖和分化、维持血脑屏障完整性以及对氧化应激和兴奋性毒性应激具有细胞保护作用，继续发挥重要作用，而这些特性在肌萎缩侧索硬化症（ALS）中都具有重要意义。ALS是一种致命疾病，其特征是运动神经元选择性丧失，但其机制尚不清楚。有证据表明，Shh可能在ALS中发挥重要作用，并且Shh信号传导在ALS中也可能受到不利影响。由于对ALS患者中Shh通路的功能状态了解甚少，因此我们试图确定ALS患者脑脊液（CSF）中的Shh蛋白水平或生物学活性是否低于对照组，以及这些指标是否与ALS疾病严重程度、疾病进展以及ALS中其他具有生物学意义的CSF分析物相关。通过酶联免疫吸附测定（ELISA）比较正常对照组（n = 13）、神经对照组（n = 12）和ALS患者（n = 9）脑脊液中的Shh水平，我们发现对照组和ALS患者的脑脊液Shh水平没有差异。然而，当使用基于体外细胞的测定方法评估脑脊液中的Shh生物学活性时，该方法将Shh活性测量为可诱导的Gli驱动的发光，我们发现，在存在外源性重组Shh或Shh激动剂嘌呤吗啡的情况下，对照组脑脊液的可诱导活性如预期显著增强，但ALS组没有，这表明ALS脑脊液样本中存在Shh信号传导抑制剂。由于嘌呤吗啡作用于Shh及其受体Patched下游的Smoothened，因此抑制作用在Smoothened下游。我们的结果还表明，虽然ALS脑脊液对Shh信号传导的抑制作用与ALS疾病特征没有显著相关性，但白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）的水平与疾病特征相关。除了在ALS脑脊液中显著升高外，这些细胞因子与疾病持续时间呈负相关，而生长分化因子11（GDF11）是ALS临床评分的一个有利预测指标。我们还发现TNF-α在体外显著抑制Shh生物学活性，这可能表明TNF-α在ALS发病机制中具有新的作用。总的来说，这是第一份证明ALS患者脑脊液中Shh信号传导受损的报告。
