IGFBP3 Colocalizes with and Regulates Hypocretin (Orexin)

BackgroundThe sleep disorder narcolepsy is caused by a vast reduction in neurons producing the hypocretin (orexin) neuropeptides. Based on the tight association with HLA, narcolepsy is believed to result from an autoimmune attack, but the cause of hypocretin cell loss is still unknown. We performed gene expression profiling in the hypothalamus to identify novel genes dysregulated in narcolepsy, as these may be the target of autoimmune attack or modulate hypocretin gene expression.
Methodology/Principal FindingsWe used microarrays to compare the transcriptome in the posterior hypothalamus of (1) narcoleptic versus control postmortem human brains and (2) transgenic mice lacking hypocretin neurons versus wild type mice. Hypocretin was the most downregulated gene in human narcolepsy brains. Among many additional candidates, only one, insulin-like growth factor binding protein 3 (IGFBP3), was downregulated in both human and mouse models and co-expressed in hypocretin neurons. Functional analysis indicated decreased hypocretin messenger RNA and peptide content, and increased sleep in transgenic mice overexpressing human IGFBP3, an effect possibly mediated through decreased hypocretin promotor activity in the presence of excessive IGFBP3. Although we found no IGFBP3 autoantibodies nor a genetic association with IGFBP3 polymorphisms in human narcolepsy, we found that an IGFBP3 polymorphism known to increase serum IGFBP3 levels was associated with lower CSF hypocretin-1 in normal individuals.
Conclusions/SignificanceComparison of the transcriptome in narcolepsy and narcolepsy model mouse brains revealed a novel dysregulated gene which colocalized in hypocretin cells. Functional analysis indicated that the identified IGFBP3 is a new regulator of hypocretin cell physiology that may be involved not only in the pathophysiology of narcolepsy, but also in the regulation of sleep in normal individuals, most notably during adolescence. Further studies are required to address the hypothesis that excessive IGFBP3 expression may initiate hypocretin cell death and cause narcolepsy.

**背景** 发作性睡病（narcolepsy）作为一种睡眠障碍，其发病机制为产生下丘脑泌素（hypocretin，又称orexin）神经肽的神经元大量减少。基于其与人类白细胞抗原（HLA）的紧密关联，发作性睡病被认为源于自身免疫攻击，但下丘脑泌素神经元丢失的具体病因迄今尚未明确。本研究通过对下丘脑进行基因表达谱分析，旨在筛选出发作性睡病患者体内失调的新基因——此类基因既可能成为自身免疫攻击的靶点，也可调控下丘脑泌素的基因表达。

**方法与主要研究结果** 本研究采用基因芯片（microarrays）技术，对比分析两组样本的下丘脑后部转录组：（1）发作性睡病患者与健康对照者的死后人类脑组织；（2）缺失下丘脑泌素神经元的转基因小鼠与野生型小鼠。在人类发作性睡病脑组织中，下丘脑泌素是下调幅度最大的基因。在众多其他候选基因中，仅胰岛素样生长因子结合蛋白3（IGFBP3, insulin-like growth factor binding protein 3）在人类与小鼠模型中均出现下调，且与下丘脑泌素神经元存在共表达现象。功能分析结果显示，过表达人类IGFBP3的转基因小鼠体内，下丘脑泌素的信使RNA（messenger RNA）与肽类含量均降低，且睡眠时长增加；该效应可能是由于过量IGFBP3存在时，下丘脑泌素的启动子活性降低所介导。尽管本研究未在人类发作性睡病患者中检测到IGFBP3自身抗体，也未发现IGFBP3基因多态性与该病存在遗传关联，但我们发现，已知可升高血清IGFBP3水平的IGFBP3基因多态性，与正常个体脑脊液（CSF, cerebrospinal fluid）中的下丘脑泌素-1（hypocretin-1）水平降低存在显著关联。

**结论与意义** 通过对比发作性睡病患者与发作性睡病模型小鼠的下丘脑后部转录组，本研究筛选出了一个与下丘脑泌素神经元共定位的新型失调基因。功能分析表明，所鉴定出的IGFBP3是调控下丘脑泌素神经元生理功能的新因子，其不仅可能参与发作性睡病的病理生理过程，还可调控正常个体的睡眠，尤其在青春期阶段的调控作用更为显著。未来仍需开展进一步研究，以验证“过量IGFBP3表达可引发下丘脑泌素神经元死亡并导致发作性睡病”这一科学假说。