Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation
Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation
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DOI：
10.1101/gad.207654.112
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发表时间：
2013-03-01
影响因子：
10.5
通讯作者：
Dougherty, Joseph D.
中科院分区：
文献类型：
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作者：
Dalal, Jasbir;Roh, Jee Hoon;Dougherty, Joseph D.
下丘脑含有下丘脑分泌素（食欲素；Hcrt）的神经元对睡眠和清醒行为的正常调节至关重要，并与进食、焦虑、抑郁和奖励有关。这些神经元的缺失导致人类和模式生物嗜睡症。然而，对这些细胞的分子表型知之甚少；以前对综合分析的尝试只有有限的敏感性或不准确。我们建立了一个Hcrt翻译核糖体亲和纯化（bacTRAP）系，用于在体内对这些神经元中所有核糖体结合转录物进行全面的翻译分析。从这个图谱中，我们鉴定出bb606000个可检测到的高于背景表达的转录本和188个在这些神经元中高度富集的转录本，包括所有已知的细胞标记物。对原位杂交数据库的盲法分析表明，其中约60%的基因以Hcrt标记样模式表达。其中15个通过双标记和显微镜证实，包括转录因子Lhx9。该基因的消融导致Hcrt神经元损失约30%，但不会对下丘脑发育造成普遍破坏。多导睡眠图和活动监测显示，这些小鼠嗜睡严重。这些数据提供了Hcrt神经元基因表达的深入和准确的轮廓，并表明Lhx9可能对这些细胞子集的规范或存活很重要。
Hypocretin (orexin; Hcrt)-containing neurons of the hypothalamus are essential for the normal regulation of sleep and wake behaviors and have been implicated in feeding, anxiety, depression, and reward. The absence of these neurons causes narcolepsy in humans and model organisms. However, little is known about the molecular phenotype of these cells; previous attempts at comprehensive profiling had only limited sensitivity or were inaccurate. We generated a Hcrt translating ribosome affinity purification (bacTRAP) line for comprehensive translational profiling of all ribosome-bound transcripts in these neurons in vivo. From this profile, we identified >6000 transcripts detectably expressed above background and 188 transcripts that are highly enriched in these neurons, including all known markers of the cells. Blinded analysis of in situ hybridization databases suggests that similar to 60% of these are expressed in a Hcrt marker-like pattern. Fifteen of these were confirmed with double labeling and microscopy, including the transcription factor Lhx9. Ablation of this gene results in a >30% loss specifically of Hcrt neurons, without a general disruption of hypothalamic development. Polysomnography and activity monitoring revealed a profound hypersomnolence in these mice. These data provide an in-depth and accurate profile of Hcrt neuron gene expression and suggest that Lhx9 may be important for specification or survival of a subset of these cells.