Translational profiling of hypocretin neurons identifies candidate molecules for sleep regulation

Abstract

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 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.[Keywords: translational profiling; hypocretin; orexin; bacTRAP; Lhx9; narcolepsy] Supplemental material is available for this article. Narcolepsy is a profound disorder of sleep regulation, characterized by excessive daytime sleepiness, sleep attacks, cataplexy, and sleep-onset REM (rapid eye movement) periods. Studies indicate that narcolepsy, especially when accompanied by cataplexy, is due to the absence of hypocretinergic neurons and signaling (Nishino et al. 2000;Thannickal et al. 2000;Bourgin et al. 2008). The hypocretins (Hcrts; also called orexins) are peptidergic neurotransmitters used by a discrete population of neurons in the lateral hypothalamus Sakurai et al. 1998). In model organisms, disruption of the gene encoding the Hcrt peptides or a Hcrt receptor (Chemelli et al. 1999;Lin et al. 1999) or, alternatively, the ablation of the neurons producing Hcrt (Gerashchenko et al. 2001;Hara et al. 2001;Zhang et al. 2007) recapitulates features of narcolepsy. In humans, there are two variations of the disorder: narcolepsy with cataplexy (NC) and narcolepsy without cataplexy (NwoC). NC is strongly linked and associated with the HLA DQB1*0602 allele, a T-cell receptor locus, and P2RY11, a receptor that can regulate immune cell survival (Hallmayer et al. 2009;Kornum et al. 2011). This strongly suggests involvement of the immune system in the etiology of NC, while the involvement in NwoC is less clear. Cold Spring Harbor Laboratory Press on May 13, 2018 -Published by genesdev.cshlp.org Downloaded from

Cell Type-Specific Expression Analysis to Identify Putative Cellular Mechanisms for Neurogenetic Disorders

“…All the mouse cell type-specific gene expression profiling experiments were conducted on a single platform, most using published translating ribosome affinity purification (TRAP) data [GEO: GSE13379 (Doyle et al, 2008);GSE30626 (Dougherty et al, 2012); GSE38668 (Dalal et al, 2013);and GSE36068 (Dougherty et al, 2013], and habenula data [GSE43164 (Görlich et al, 2013)], except for retinal cell data (Corbo et al, 2007). Cell-specific gene lists for the cell types ( Fig.…”

“…To test this approach, we combined our analysis with a transcriptomic study of narcolepsy (Honda et al, 2009). Narcolepsy can be caused by a loss of Hcrt-expressing neurons (Peyron et al, 2000), which we have profiled (Dalal et al, 2013). Microarray data from mice where these neurons have been ablated identified 63 downregulated genes, which were sufficient to specifically indicate a loss of Hcrt neurons (Fig.…”

“…First, we merged the data from our surveys of cell type-specific gene expression from the mouse brain (Doyle et al, 2008;Dougherty et al, 2012Dalal et al, 2013;Görlich et al, 2013). For the purposes of the specific hypotheses cited below, we have also incorporated published data profiling specific retinal cell types on the same platform (Corbo et al, 2007).…”

“…In previous work, we generated bacTRAP mouse lines profiling the expression of protein-coding genes in dozens of targeted cell types (Doyle et al, 2008;Dougherty et al, 2012Dalal et al, 2013;Xu et al, 2013). Here, we combine these two sources of information and test the hypothesis that the SE of genes can guide us toward identifying the cellular disruptions mediating disorders.…”

Evolutionarily conserved regulation of hypocretin neuron specification by Lhx9

“…Despite the importance of the Hcrt system, little is known about the developmental processes that give rise to Hcrt neurons. A recent study found that mice lacking the LIM domain homeobox transcription factor Lhx9 had fewer Hcrt neurons (Dalal et al, 2013), suggesting that Lhx9 is required to specify a subset of Hcrt neurons. However, overexpression of Lhx9 in adult mice or in a mouse neuroblastoma cell line had no effect on Hcrt cell number or expression.…”