Exacerbated neuronal ceroid lipofuscinosos phenotype in Cln1/5 double knock-out mice

Both CLN1 and CLN5 deficiency leads to severe neurodegenerative diseases of childhood, known as neuronal ceroid lipofuscinoses (NCL). The broadly similar phenotypes of NCL mouse models, and the potential for interactions between NCL proteins, raise the possibility of shared or converging disease mechanisms. To begin addressing these issues we have developed a novel mouse model lacking both Cln1 and Cln5 genes. These Cln1/5 double knock-out (Cln1/5 dko) mice were fertile, showing a slight decrease in expected Mendelian breeding ratios, as well as impaired embryoid body formation of induced pluripotent stem cells derived from Cln1/5 dko fibroblasts. Typical manifestations of the NCL diseases, seizures and motor dysfunction, were detected at the age of 3 months, earlier than in either single knock-out mouse. Pathological analyses revealed a similar exacerbation and earlier onset of disease in Cln1/5 dko mice, which exhibit a pronounced accumulation of autofluorescent storage material. Cortical demyelination and more pronounced glial activation in cortical and thalamic regions was followed by cortical neuron loss. Alterations in lipid metabolism in Cln1/5 dko showed specifically an increase in serum phospholipid transfer protein (PLTP) activity. Finally, gene expression profiling of Cln1/5 dko cortex revealed defects in myelination and immune response pathways, with a prominent downregulation of alpha-synuclein in Cln1/5 dko mouse brains. The simultaneous loss of both Cln1 and Cln5 genes may enhance the typical pathological phenotypes of these mice by disrupting down shared or convergent pathogenic pathways, which may potentially include interactions of CLN1 and CLN5. Basic characterization of Cln1/5 double knock-out mouse model. Aim was to find possible differentially expressed genes and up-or downregulated pathways in Cln1/5 double knock-out vs. wild-type mouse cortex. Total RNA isolated from 1 month old Cln1-/-/Cln5-/- mouse cortex.CLN1与CLN5缺陷均可引发儿童期重型神经退行性疾病——神经元蜡样脂褐质沉积症（neuronal ceroid lipofuscinoses, NCL）。NCL小鼠模型的表型高度相似，且NCL蛋白间存在相互作用的潜在可能，提示二者可能共享或趋同的疾病发病机制。为探究上述问题，本研究构建了同时敲除Cln1与Cln5基因的新型小鼠模型。该Cln1/5双敲除（Cln1/5 dko）小鼠可正常繁育，但孟德尔育种预期比例略有降低；其成纤维细胞来源的诱导多能干细胞（induced pluripotent stem cells）的拟胚体（embryoid body）形成能力受损。NCL疾病的典型表现——癫痫发作与运动功能障碍，于该模型小鼠3月龄时即可被检测到，发病早于任一单基因敲除小鼠模型。病理分析结果显示，Cln1/5 dko小鼠的疾病表型显著加重且发病提前，其体内可见明显的自体荧光储存物质蓄积。随后该模型小鼠出现皮层脱髓鞘病变，皮层与丘脑区域的胶质细胞激活程度显著增强，继而发生皮层神经元丢失。Cln1/5 dko小鼠的脂质代谢异常表现为血清磷脂转运蛋白（phospholipid transfer protein, PLTP）活性特异性升高。最后，对Cln1/5 dko小鼠皮层的基因表达谱分析显示，其髓鞘形成与免疫应答通路存在功能缺陷，且小鼠脑内α-突触核蛋白的表达量显著下调。同时缺失Cln1与Cln5基因，可能通过破坏共享或趋同的致病通路（该通路或涉及CLN1与CLN5的相互作用），加重该模型小鼠的典型病理表型。本研究对Cln1/5双敲除小鼠模型进行了基础表征，旨在筛选Cln1/5双敲除与野生型小鼠皮层组织间的差异表达基因，并探究二者间上调或下调的信号通路。实验所用样品为1月龄Cln1-/-/Cln5-/-小鼠皮层组织中提取的总RNA。