Data Sheet 1_Tmem45b modulates itch via endoplasmic reticulum calcium regulation.docx

ObjectiveThis study aimed to investigate the role of Tmem45b, a gene expressed in itch-associated Dorsal root ganglion (DRG) neurons, in the regulation of itch sensation.
MethodsThe expression of Tmem45b was examined in DRG neurons. These neurons included Nppb-, Mrgpra3-, and Mrgprd-positive subtypes, which are known to mediate itch. Behavioral response to various pruritogens including β-alanine, chloroquine, histamine, serotonin, and N-met-LTC4 were assessed on Mrgprd-cre::Tmem45bflox/flox conditional knockout (cKO) mice. Chronic itch was evaluated using both atopic dermatitis-like and dry skin-like mouse models. To investigate intracellular calcium dynamics, calcium imaging was performed on dissociated DRG neurons. Additionally, bulk RNA-seq was conducted on DRG from Tmem45b cKO mice to assess transcriptomic changes. Serca1 expression and the calcium storage capacity of the endoplasmic reticulum (ER) were analyzed following Tmem45b deletion.
ResultsTmem45b was found to be expressed in itch-associated DRG neurons. In Tmem45b cKO mice, scratching behavior was reduced in response to β-alanine but increased in response to chloroquine. Notably, chronic itch was alleviated in Tmem45b-deficient mice. Calcium imaging revealed that Tmem45b cKO impaired calcium responses to β-alanine and allyl isothiocyanate, but not to chloroquine. Mechanistically, Tmem45b deficiency led to a significant downregulation of Serca1, reducing ER calcium storage capacity. Pharmacological inhibition of Serca1 in DRG neurons similarly suppressed intracellular calcium release in response to β-alanine and chloroquine.
ConclusionTmem45b plays a critical role in nonhistaminergic itch by regulating ER calcium homeostasis through Serca1. Its deficiency reduces itch behavior and impairs calcium signaling in DRG neurons, suggesting that Tmem45b is a potential therapeutic target for chronic itch.

研究目的
本研究旨在探究在瘙痒相关背根神经节（Dorsal root ganglion, DRG）神经元中表达的Tmem45b基因在瘙痒感觉调控中的作用。
研究方法
研究人员检测了DRG神经元中Tmem45b的表达情况，所检测的神经元包括已知可介导瘙痒的Nppb阳性、Mrgpra3阳性以及Mrgprd阳性亚型。针对Mrgprd-cre::Tmem45bflox/flox条件性敲除（conditional knockout, cKO）小鼠，研究人员评估了其对β-丙氨酸、氯喹、组胺、5-羟色胺以及N-甲基-LTC4等多种致痒剂的行为学反应。本研究通过特应性皮炎样与干性皮肤样小鼠模型，对慢性瘙痒进行评价。为探究细胞内钙动力学，研究人员对解离后的DRG神经元开展钙成像实验。此外，对Tmem45b条件性敲除小鼠的DRG组织进行批量RNA测序（bulk RNA-seq），以分析转录组变化。在Tmem45b缺失后，研究人员还分析了Serca1的表达水平以及内质网（endoplasmic reticulum, ER）的钙储存能力。
研究结果
研究发现Tmem45b在瘙痒相关DRG神经元中存在表达。在Tmem45b条件性敲除小鼠中，其对β-丙氨酸诱导的搔抓行为显著减少，但对氯喹诱导的搔抓行为则有所增强。值得注意的是，Tmem45b缺陷小鼠的慢性瘙痒症状得到明显缓解。钙成像实验结果显示，Tmem45b条件性敲除会削弱DRG神经元对β-丙氨酸和异硫氰酸烯丙酯的钙响应，但不影响其对氯喹的钙响应。机制层面分析表明，Tmem45b缺陷会导致Serca1的表达显著下调，进而降低内质网的钙储存能力。对DRG神经元进行Serca1的药理学抑制，同样会抑制其对β-丙氨酸和氯喹的细胞内钙释放反应。
研究结论
Tmem45b通过调控Serca1介导的内质网钙稳态，在非组胺能瘙痒中发挥关键作用。其缺失会减少搔抓行为并损害DRG神经元的钙信号通路，提示Tmem45b是慢性瘙痒潜在的治疗靶点。