Göcz Balázs, Rumpler Éva, Szentkirályi-Tóth Soma, Skrapits Katalin, Takács Szabolcs, Sárvári Miklós, Farkas Imre, Póliska Szilárd, Hrabovszky Erik
Laboratory of Reproductive Neurobiology, HUN-REN Institute of Experimental Medicine, Budapest, Hungary.
Laboratory of Reproductive Neurobiology, HUN-REN Institute of Experimental Medicine, Budapest, Hungary.
J Biol Chem. 2025 Feb;301(2):108150. doi: 10.1016/j.jbc.2024.108150. Epub 2024 Dec 28.
We developed a versatile 'IHC/LCM-Seq' method for spatial transcriptomics of immunohistochemically detected neurons collected with laser-capture microdissection (LCM). IHC/LCM-Seq uses aluminon and polyvinyl sulfonic acid for inventive RNA-preserving strategies to maintain RNA integrity in free-floating sections of 4% formaldehyde-fixed brains. To validate IHC/LCM-Seq, we first immunostained and harvested striatal cholinergic interneurons with LCM. RNA preparations were subjected to random primer-based cDNA library preparation and bulk sequencing on the NextSeq Illumina platform. IHC/LCM-Seq detected ∼16,000 transcripts, reaching the sensitivity of a reference 'LCM-Seq method' developed for fluorescently tagged neurons microdissected from lightly formaldehyde-fixed and slide-mounted brain sections of transgenic mice. We successfully used the new IHC/LCM-Seq approach to provide unprecedented insight into the transcriptome of immunohistochemically detected gonadotropin-releasing hormone (GnRH) neurons regulating reproduction. The ∼13,000 to 14,000 transcripts identified in GnRH neurons of adult male rats and mice encoded 28 proteins implicated previously in human infertility, 35 neuropeptides, 34 nuclear receptors, and 164 G protein-coupled receptors. Functional experiments using slice electrophysiology established that the heavy Ntsr2 expression conveys a strong excitatory action of neurotensin on GnRH neurons. As an unexpected species difference, we found that GnRH neurons exclusively expressed estrogen receptor-β in rats and against the current consensus, the alpha estrogen receptor isoform in mice. The IHC/LCM-Seq technique we are reporting is a highly sensitive and accurate bulk sequencing approach to characterize the transcriptome landscape of immunohistochemically labeled neurons, including neuroendocrine GnRH cells. This method is readily applicable to any species, opening new perspectives also for future studies of the post mortem human brain.
我们开发了一种通用的“免疫组化/激光捕获显微切割测序（IHC/LCM-Seq）”方法，用于对通过激光捕获显微切割（LCM）收集的免疫组化检测神经元进行空间转录组学分析。IHC/LCM-Seq使用铝试剂和聚乙烯磺酸进行创新的RNA保存策略，以在4%甲醛固定脑的游离切片中维持RNA完整性。为了验证IHC/LCM-Seq，我们首先用LCM对纹状体胆碱能中间神经元进行免疫染色和收获。RNA制剂进行基于随机引物的cDNA文库制备，并在Illumina NextSeq平台上进行批量测序。IHC/LCM-Seq检测到约16,000个转录本，达到了为从轻度甲醛固定和载玻片安装的转基因小鼠脑切片中显微切割的荧光标记神经元开发的参考“LCM-Seq方法”的灵敏度。我们成功地使用新的IHC/LCM-Seq方法，以前所未有的方式深入了解调节生殖的免疫组化检测促性腺激素释放激素（GnRH）神经元的转录组。在成年雄性大鼠和小鼠的GnRH神经元中鉴定出的约13,000至14,000个转录本编码了28种先前与人类不孕症有关的蛋白质、35种神经肽、34种核受体和164种G蛋白偶联受体。使用切片电生理学的功能实验表明，重Ntsr2表达传递神经降压素对GnRH神经元的强烈兴奋作用。作为一个意外的物种差异，我们发现GnRH神经元在大鼠中仅表达雌激素受体-β，与当前共识相反，在小鼠中表达α雌激素受体异构体。我们报道的IHC/LCM-Seq技术是一种高度灵敏和准确的批量测序方法，用于表征免疫组化标记神经元（包括神经内分泌GnRH细胞）的转录组景观。这种方法很容易应用于任何物种，也为未来人类死后大脑的研究开辟了新的前景。
