Hedgehog Interaction Protein Acts in the Habenula to Regulate Nicotine Intake

Medial habenular (mHb) cholinergic neurons that project to the interpeduncular nucleus (IPn) regulate aversive behavioral responses to nicotine that protect against tobacco addiction. Little is known about the nicotine-evoked cellular or molecular adaptations in these neurons that influence the development of the smoking habit. Using in vivo calcium imaging and single-cell RNA sequencing, we show that a dose of nicotine that stimulates mHb neural activity evokes robust transcriptional plasticity in neuronal and non-neuronal cells in the mHb, including upregulated expression of Hedgehog-interacting protein (HHIP) in putative cholinergic neurons. Allelic variation in HHIP confers risk for smoking-related diseases including chronic obstructive pulmonary disease and lung cancer, but underlying mechanisms of action are unclear. Using Translating Ribosome Affinity Purification (TRAP) sequencing and RNAscope, we confirmed that Hhip transcripts are highly enriched in mHb cholinergic neurons. HHIP mutant mice exhibit hundreds of differentially expressed transcripts in the mHb and perturbed transcriptional responses to nicotine. Moreover, acute in vivo CRISPR/Cas9-mediated genomic cleavage of mHb Hhip attenuated noxious responses to nicotine and increased intravenous nicotine self-administration behavior in mice. In vitro knockdown of Hhip reduces intracellular calcium release to nicotine and increases Gli activity. These findings suggest that HHIP acts in the mHb to regulate nicotine intake and that HHIP alleles may increase vulnerability to smoking-related diseases by modulating mHb signaling and enhancing the addictive properties of tobacco. Overall design: HHIP mutant x WT. Saline x Nicotine. Medial Habenular tissue.

投射至脚间核（interpeduncular nucleus，IPn）的内侧缰核（medial habenular，mHb）胆碱能神经元，可调控针对尼古丁的厌恶行为反应，从而对烟草成瘾起到防御作用。目前对于这些神经元中由尼古丁诱发的、可影响吸烟习惯形成的细胞与分子适应性改变，尚缺乏充分研究。本研究通过活体钙成像与单细胞RNA测序技术，发现可激活内侧缰核神经元活动的尼古丁剂量，能在内侧缰核的神经元与非神经元细胞中诱发显著的转录可塑性，包括在推定胆碱能神经元中上调刺猬蛋白相互作用蛋白（Hedgehog-interacting protein，HHIP）的表达。HHIP的等位基因变异会增加吸烟相关疾病（包括慢性阻塞性肺疾病与肺癌）的患病风险，但其具体作用机制尚不明确。本研究通过翻译核糖体亲和纯化（Translating Ribosome Affinity Purification，TRAP）测序与RNAscope技术，证实Hhip转录本在内侧缰核胆碱能神经元中高度富集。HHIP突变小鼠的内侧缰核中存在数百个差异表达转录本，且其对尼古丁的转录应答出现异常。此外，通过活体急性CRISPR/Cas9介导的内侧缰核Hhip基因组切割，可减弱小鼠对尼古丁的厌恶反应，并提升其静脉尼古丁自身给药行为。体外敲低Hhip可减少尼古丁诱导的细胞内钙释放，并增强Gli通路活性。上述研究结果表明，HHIP可在内侧缰核中调控尼古丁摄入，且HHIP等位基因可能通过调节内侧缰核信号传导、增强烟草的成瘾特性，提升个体罹患吸烟相关疾病的易感性。实验整体设计：HHIP突变小鼠与野生型（wild type，WT）对照；生理盐水与尼古丁处理；取材内侧缰核组织。