Guo Qi, Kouyama-Suzuki Emi, Shirai Yoshinori, Tabuchi Katsuhiko
Department of Molecular and Cellular Physiology, Shinshu University School of Medicine, Matsumoto 390-8621, Japan.
Department of NeuroHealth Innovation, Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto 390-8621, Japan.
Cells. 2025 May 20;14(10):750. doi: 10.3390/cells14100750.
Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a severe neurodevelopmental disorder caused by a deficiency in the X-linked gene calcium/calmodulin-dependent serine protein kinase (CASK). A better understanding of the role of CASK in the pathophysiology of neurodevelopmental disorders may provide insights into novel therapeutic and diagnostic strategies for MICPCH syndrome and other neurodegenerative diseases. To investigate this, we generated CASK knockout (KO) cerebellar granule (CG) cell culture from CASK floxed (CASK) mice by infecting lentiviruses expressing codon-improved Cre recombinase (iCre). We performed RNA-sequencing (RNA-seq) on these cells and found that CASK-KO CG cells underwent apoptosis by activating intracellular Jun N-terminal kinase (JNK) signaling and upregulating reactive oxygen species (ROS)-related gene expression. We also performed mouse gait analysis and limb clasping behavior experiments on trans-heterozygous CASK-KO and Hprt-eGFP (CASK Hprt) mice. The CASK Hprt mice exhibited cerebellar ataxic phenotypes as judged by the scores of these experiments compared to the CASK wild-type control (CASK Hprt) mice. Interestingly, the administration of the JNK inhibitor, JNK-IN-8, in CASK-KO CG cell cultures increased CG cell survival by reducing ROS generation. Moreover, injection of JNK-IN-8 into the cerebellum of CASK Hprt mice suppressed CG cell death and alleviated cerebellar ataxic phenotypes in vivo. In conclusion, JNK-IN-8 suppresses the cell death and activation of the ROS pathway in CASK-KO CG cells in both in vitro and in vivo models, suggesting its potential as a therapeutic strategy for cerebellar neurodegeneration in MICPCH syndrome.
小头畸形伴脑桥和小脑发育不全（MICPCH）综合征是一种严重的神经发育障碍，由X连锁基因钙/钙调蛋白依赖性丝氨酸蛋白激酶（CASK）缺陷引起。更好地了解CASK在神经发育障碍病理生理学中的作用，可能为MICPCH综合征和其他神经退行性疾病的新型治疗和诊断策略提供见解。为了对此进行研究，我们通过感染表达密码子优化的Cre重组酶（iCre）的慢病毒，从CASK条件性敲除（floxed，CASK）小鼠中生成了CASK基因敲除（KO）的小脑颗粒（CG）细胞培养物。我们对这些细胞进行了RNA测序（RNA-seq），发现CASK-KO CG细胞通过激活细胞内的Jun N端激酶（JNK）信号通路并上调活性氧（ROS）相关基因的表达而发生凋亡。我们还对反式杂合CASK-KO和Hprt-eGFP（CASK Hprt）小鼠进行了小鼠步态分析和肢体紧握行为实验。与CASK野生型对照（CASK Hprt）小鼠相比，根据这些实验的评分判断，CASK Hprt小鼠表现出小脑共济失调的表型。有趣的是，在CASK-KO CG细胞培养物中施用JNK抑制剂JNK-IN-8，通过减少ROS生成提高了CG细胞的存活率。此外，将JNK-IN-8注射到CASK Hprt小鼠的小脑中，在体内抑制了CG细胞死亡并减轻了小脑共济失调的表型。总之，JNK-IN-8在体外和体内模型中均抑制了CASK-KO CG细胞中的细胞死亡和ROS途径的激活，表明其作为MICPCH综合征中小脑神经退行性变治疗策略的潜力。
