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靶向单细胞表达谱分析可识别睡眠与代谢状态的整合因子。

Targeted single cell expression profiling identifies integrators of sleep and metabolic state.

作者信息

Shih Meng-Fu Maxwell, Zhang Jiwei, Brown Elizabeth B, Dubnau Joshua, Keene Alex C

机构信息

Dept of Anesthesiology, Stony Brook School of Medicine, Stony Brook NY, 11794.

Department of Biology, Texas A&M University, College Station, TX 77840.

出版信息

bioRxiv. 2024 Sep 27:2024.09.25.614841. doi: 10.1101/2024.09.25.614841.

Abstract

Animals modulate sleep in accordance with their internal and external environments. Metabolic cues are particularly potent regulators of sleep, allowing animals to alter their sleep timing and amount depending on food availability and foraging duration. The fruit fly, , suppresses sleep in response to acute food deprivation, presumably to forage for food. This process is dependent on a single pair of Lateral Horn Leucokinin (LHLK) neurons, that secrete the neuropeptide Leucokinin. These neurons signal to insulin producing cells and suppress sleep under periods of starvation. The identification of individual neurons that modulate sleep-metabolism interactions provides the opportunity to examine the cellular changes associated with sleep modulation. Here, we use single-cell sequencing of LHLK neurons to examine the transcriptional responses to starvation. We validate that a Patch-seq approach selectively isolates RNA from individual LHLK neurons. Single-cell CEL-Seq comparisons of LHLK neurons between fed and 24-hr starved flies identified 24 genes that are differentially expressed in accordance with starvation state. In total, 12 upregulated genes and 12 downregulated genes were identified. Gene-ontology analysis showed an enrichment for , a family of anti-microbial peptides, along with several transcripts with diverse roles in regulating cellular function. Targeted knockdown of differentially expressed genes identified multiple genes that function within LHLK neurons to regulate sleep-metabolism interactions. Functionally validated genes include an essential role for the E3 ubiquitin Ligase , the sorbitol dehydrogenase , as well as and in starvation-induced sleep suppression. Taken together, these findings provide a pipeline for identifying novel regulators of sleep-metabolism interactions within individual neurons.

摘要

动物会根据其内部和外部环境来调节睡眠。代谢线索是睡眠的特别有力的调节因素,使动物能够根据食物供应情况和觅食持续时间改变其睡眠时间和睡眠量。果蝇在急性食物剥夺时会抑制睡眠,大概是为了觅食。这个过程依赖于一对分泌神经肽促胃液素释放肽的外侧角促胃液素释放肽(LHLK)神经元。这些神经元向产生胰岛素的细胞发出信号,并在饥饿期间抑制睡眠。对调节睡眠 - 代谢相互作用的单个神经元的识别,为研究与睡眠调节相关的细胞变化提供了机会。在这里,我们使用LHLK神经元的单细胞测序来研究对饥饿的转录反应。我们验证了一种膜片钳测序方法能从单个LHLK神经元中选择性地分离RNA。对喂食和饥饿24小时的果蝇的LHLK神经元进行单细胞CEL-Seq比较,确定了24个根据饥饿状态差异表达的基因。总共鉴定出12个上调基因和12个下调基因。基因本体分析显示,抗菌肽家族以及在调节细胞功能中具有多种作用的几个转录本有富集。对差异表达基因的靶向敲低确定了多个在LHLK神经元内发挥作用以调节睡眠 - 代谢相互作用的基因。功能验证的基因包括E3泛素连接酶、山梨醇脱氢酶以及在饥饿诱导的睡眠抑制中的重要作用。综上所述,这些发现提供了一个用于识别单个神经元内睡眠 - 代谢相互作用新调节因子的途径。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/bf10/11463630/7784039db5e5/nihpp-2024.09.25.614841v1-f0001.jpg

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