Wæver Sofia Lyng, Haanes Kristian Agmund
Sensory Biology Unit, Translational Research Center, Rigshospitalet, Glostrup, Denmark.
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
J Headache Pain. 2025 Jan 14;26(1):11. doi: 10.1186/s10194-024-01945-6.
The neuropeptide calcitonin gene-related peptide (CGRP) has been established to be a key signaling molecule in migraine, but little is known about the differences between the two isoforms: αCGRP and βCGRP. Previous studies have been hampered by their close similarity, making the development of specific antibodies nearly impossible. In this study we sought to test the hypothesis that αCGRP and βCGRP localize differently within the neurons of the mouse trigeminal ganglion (TG), using αCGRP knock out (KO) animals.
We applied immunohistochemistry (IHC) on 15 TGs from three different genotypes of mice; wild type (WT) αCGRP heterozygote (Het) and αCGRP KOs, with a primary antibody targeting the mature neuropeptide sequence of both αCGRP and βCGRP. Subsequently, the localization patterns of the two isoforms were analyzed. Furthermore, similar IHCs were produced in KO animals after being treated with monoclonal CGRP antibodies to study the origin of the observed CGRP. Additional IHCs were conducted in KO and WT mice to locate CGRP sorting peptides within neuronal cell bodies. Lastly, bioinformatical analyses of the primary, secondary, and tertiary structure of the two isoforms were conducted.
The IHC showed that the key isoform localized within the axons of the mouse TG neurons, is αCGRP and not βCGRP. Furthermore, differences in intensities indicate that the model used in this study successfully knocks out αCGRP. We further categorized the localization patterns of CGRP in neuronal cell bodies in the TG and found using bioinformatic analyses that differences in localization might be explained by intracellular peptide sorting. IHC following injections with monoclonal CGRP antibodies in KO mice ruled out the possibility that the βCGRP observed in trigeminal neurons had peripheral origins. This conclusion was enhanced by IHC experiments which showed the presence of CGRP co-localizing sorting peptides in KO mice.
Our data show that mainly αCGRP and not βCGRP locate within the axons of the mouse TG neurons. The βCGRP observed within the TG neuronal cell bodies is synthesized intracellularly and not taken up from the environment. Furthermore, the isoforms appear to be sorted differentially into secretory vesicles in the cell bodies of TG neurons.
神经肽降钙素基因相关肽（CGRP）已被确认为偏头痛中的关键信号分子，但对于两种亚型：αCGRP和βCGRP之间的差异知之甚少。先前的研究因它们的高度相似性而受阻，使得几乎不可能开发出特异性抗体。在本研究中，我们试图使用αCGRP基因敲除（KO）动物来验证αCGRP和βCGRP在小鼠三叉神经节（TG）神经元内定位不同的假设。
我们对来自三种不同基因型小鼠（野生型（WT）、αCGRP杂合子（Het）和αCGRP基因敲除小鼠）的15个三叉神经节进行免疫组织化学（IHC）检测，使用针对αCGRP和βCGRP成熟神经肽序列的一抗。随后，分析这两种亚型的定位模式。此外，在用单克隆CGRP抗体处理后的基因敲除动物中进行类似的免疫组织化学检测，以研究观察到的CGRP的来源。在基因敲除小鼠和野生型小鼠中进行额外的免疫组织化学检测，以定位神经元细胞体内的CGRP分选肽。最后，对这两种亚型的一级、二级和三级结构进行生物信息学分析。
免疫组织化学显示，在小鼠三叉神经节神经元轴突内定位的关键亚型是αCGRP而非βCGRP。此外，强度差异表明本研究中使用的模型成功敲除了αCGRP。我们进一步对三叉神经节中神经元细胞体内CGRP的定位模式进行了分类，并通过生物信息学分析发现，定位差异可能由细胞内肽分选来解释。在基因敲除小鼠中注射单克隆CGRP抗体后的免疫组织化学检测排除了在三叉神经元中观察到的βCGRP具有外周来源的可能性。基因敲除小鼠中存在CGRP共定位分选肽的免疫组织化学实验进一步证实了这一结论。
我们的数据表明，主要是αCGRP而非βCGRP定位于小鼠三叉神经节神经元的轴突内。在三叉神经节神经元细胞体内观察到的βCGRP是在细胞内合成的，而非从环境中摄取。此外，这两种亚型在三叉神经节神经元细胞体内似乎被不同地分选到分泌小泡中。
