Fu Rong, Ding Man, Yin Tong, Zheng Linlin, Liu Yue, Yu Hang, Zhou Rumeng, Lu Zuneng
Department of Neurology, Renmin Hospital of Wuhan University, Wuhan, 430060, Hu Bei Province, China.
Sci Rep. 2025 Jul 1;15(1):20683. doi: 10.1038/s41598-025-06398-8.
Ankyrin repeat and FYVE domain containing 1 (ANKFY1) is an indispensable protein in the development of cerebellar Purkinje cells. Our preliminary study revealed that its absence caused progressive spastic ataxia, accompanied by the loss of Purkinje cells in mice. Here, we generated Ankfy1-floxed (Ankfy1) mice, in which conditional inactivation of the Ankfy1 gene was achieved specifically in cerebellar Purkinje cells via crossing with a transgenic mouse strain expressing Cre recombinase under the regulatory control of the Purkinje cell protein 2 (PCP2) promoter. We employed data-independent acquisition (DIA) mass spectrometry to compare the protein expression profiles in cerebellar samples. The samples were obtained from two groups of male mice. The first group consisted of three Pcp2-Cre; Ankfy1 male mice, where the Ankfy1 gene was conditionally knocked out in Purkinje cells, and these mice were designated as the conditional knockout (CKO) group. The second group included three Cre-negative; Ankfy1 littermate male mice served as the wild-type (WT) control group. The results identified 69 (45 upregulated and 24 downregulated) differentially expressed proteins (DEPs) in CKO vs. WT male mice with a 1.5-fold change. Enrichment analyses of these DEPs based on the Gene Ontology (GO), Orthologous Groups of Proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases revealed functional clusters associated with neuronal cell morphogenesis, extracellular structures, regulation of Rho-GTPase, calcium signaling pathway, etc. Itgb2, which was upregulated in CKO mice, was the top hub gene according to protein-protein interaction (PPI) analysis. We selected seven interesting differentially expressed genes (Arhgdib, Impa2, Pcp2, Pcp4, Ppp1r17, Rhobtb2, and Cdc123) for further validation. Arhgdib and Imp2a expression was increased in the cerebellum of CKO male and female mice, and Pcp2 and Pcp4 expression was decreased. Western blotting and immunofluorescence verified the upregulation of ARHGDIB and the downregulation of PCP2 in the cerebellum. Our proteomic analysis of conditional Ankfy1 knockout mice may guide future research to help develop treatments for progressive spastic ataxia.
含锚蛋白重复序列和FYVE结构域蛋白1（ANKFY1）是小脑浦肯野细胞发育中不可或缺的蛋白质。我们的初步研究表明，ANKFY1缺失会导致进行性痉挛性共济失调，并伴有小鼠浦肯野细胞的丢失。在此，我们构建了Ankfy1基因条件性敲除（Ankfy1）小鼠，通过与在浦肯野细胞蛋白2（PCP2）启动子调控下表达Cre重组酶的转基因小鼠品系杂交，实现Ankfy1基因在小脑浦肯野细胞中的特异性条件性失活。我们采用数据非依赖采集（DIA）质谱法比较小脑样本中的蛋白质表达谱。样本取自两组雄性小鼠。第一组由三只Pcp2-Cre; Ankfy1雄性小鼠组成，其中Ankfy1基因在浦肯野细胞中被条件性敲除，这些小鼠被指定为条件性敲除（CKO）组。第二组包括三只Cre阴性的; Ankfy1同窝雄性小鼠作为野生型（WT）对照组。结果在CKO与WT雄性小鼠中鉴定出69种（45种上调和24种下调）差异表达蛋白（DEP），变化倍数为1.5倍。基于基因本体论（GO）、蛋白质直系同源簇（COG）和京都基因与基因组百科全书（KEGG）数据库对这些DEP进行的富集分析揭示了与神经元细胞形态发生、细胞外结构、Rho-GTPase调节、钙信号通路等相关的功能簇。根据蛋白质-蛋白质相互作用（PPI）分析，在CKO小鼠中上调的Itgb2是核心枢纽基因。我们选择了七个有趣的差异表达基因（Arhgdib、Impa2、Pcp2、Pcp4、Ppp1r17、Rhobtb2和Cdc123）进行进一步验证。Arhgdib和Imp2a在CKO雄性和雌性小鼠小脑中的表达增加，而Pcp2和Pcp4的表达降低。蛋白质免疫印迹和免疫荧光验证了小脑中ARHGDIB的上调和PCP2的下调。我们对条件性Ankfy1敲除小鼠的蛋白质组学分析可能会为未来的研究提供指导，以帮助开发进行性痉挛性共济失调的治疗方法。
