Deciphering the role of importin alpha in mammalian central neurons - Hippocampal gene expression profiling in importin alpha5-deleted mice

Introduction: The mechanisms governing synaptonuclear transport, and the transcriptional pathways that are at the core of neuronal disorders are poorly understood. The importin family of nuclear import factors has pivotal roles in such pathways, due to their involvement in intracellular transport from both synapse to soma and cytoplasm to nucleus. Mammals express six different isoforms of importin α, which bind an Importin β to form transport complexes for specific cargoes. Methods: We addressed the roles of importin α isoforms in mammalian CNS by evaluation of a systematic series of importin α knockout mice. A comprehensive battery of behavioral tests was used to investigate spontaneous and novelty-induced locomotion, basal ganglia function, motor coordination, neuromuscular integration, anxiety-related behaviors and memory. Subsequent analyses using acute-brain slice electrophysiology, RNA-seq, bioinformatics, imaging and pharmacology was used in order to delineate the contribution of genes and signaling pathways involved in the identified phenotype and screen for new therapeutic approaches. Results and Discussion: During the course of comprehensive behavioral profiling of importin α mouse mutants, we identified an importin α knockout (KO) mouse with significantly reduced anxiety levels. Electrophysiological recordings in acute hippocampal slices showed a reduced short-term and presynaptic plasticity (paired-pulse facilitation deficit, lower PTP), while LTP was not affected in these mutants. We then performed transcriptional profiling (RNA-seq) from hippocampal extracts of wild type versus mutant animals after exposure to an anxiogenic drug. Computational analysis revealed differences in the expression of genes and the involvement of transcription factors. Drugs that activate LXR or the Sphingosine 1 phosphate receptor have robust anxiolytic effects, while a Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse. Thus, importin α5 influences anxiety by regulating nuclear import in neurons. RNA-seq performed on: 4 experimental groups, 3 animals per each vehicle group and replicate, 3 replicates of the entire set - Exception in the FG7142-treated Importin α5-/- group (n=2) where 1 animal was identified outlier and discarded from the analysis

引言：调控突触核运输（synaptonuclear transport）以及作为神经疾病核心的转录通路的相关机制目前仍未得到充分阐明。核输入因子家族中的输入蛋白（importin）在这类通路中发挥关键作用，因其参与从突触到胞体以及从细胞质到细胞核的细胞内运输过程。哺乳动物体内存在6种不同的输入蛋白α亚型，它们可结合输入蛋白β以形成针对特定转运底物的转运复合物。
方法：本研究通过构建一系列系统性的输入蛋白α基因敲除小鼠，探究其在哺乳动物中枢神经系统（Central Nervous System, CNS）中的功能。我们采用一套全面的行为学测试组合，以评估自发活动与新异刺激诱导的运动能力、基底节功能、运动协调能力、神经肌肉整合功能、焦虑相关行为以及记忆能力。后续我们通过急性脑片电生理学、RNA测序（RNA-seq）、生物信息学、成像技术以及药理学分析，明确参与所观测表型的基因与信号通路的作用，并筛选潜在的新型治疗策略。
结果与讨论：在对输入蛋白α基因敲除小鼠突变体进行全面行为谱分析的过程中，我们发现一只输入蛋白α基因敲除（KO）小鼠的焦虑水平显著降低。对急性海马脑片的电生理记录显示，该突变体的短期突触可塑性与突触前可塑性均出现受损，表现为配对脉冲易化缺陷、强直后增强（Post-tetanic Potentiation, PTP）水平降低，而长时程增强（Long-term Potentiation, LTP）未受影响。随后我们将野生型与突变型小鼠暴露于致焦虑药物后，提取其海马组织并进行转录组分析（RNA-seq）。计算分析结果显示，两组小鼠的基因表达存在差异，且转录因子的调控作用也有所不同。激活肝X受体（Liver X Receptor, LXR）或1-磷酸鞘氨醇受体（Sphingosine 1-phosphate Receptor）的药物均具有显著的抗焦虑作用，而鞘氨醇激酶1（Sphingosine Kinase 1, Sphk1）抑制剂可逆转输入蛋白α5基因敲除小鼠的抗焦虑表型。综上，输入蛋白α5通过调控神经元内的核输入过程影响焦虑行为。本研究的RNA测序（RNA-seq）样本设置如下：共4个实验组，溶剂对照组每组各3只动物，完整数据集共设置3次生物学重复；FG7142处理的输入蛋白α5基因敲除（α5-/-）组为例外，该组初始样本量为2，其中1只动物被鉴定为异常值并从分析中剔除。