Leggieri Adele, García-González Judit, Torres-Perez Jose V, Havelange William, Hosseinian Saeedeh, Mech Aleksandra M, Keatinge Marcus, Busch-Nentwich Elisabeth M, Brennan Caroline H
School of Biological and Behavioural Sciences, Queen Mary University of London, London, United Kingdom.
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, United States.
Front Neurosci. 2022 Feb 8;16:794653. doi: 10.3389/fnins.2022.794653. eCollection 2022.
Ankyrin repeat and kinase domain containing 1 (ANKK1) is a member of the receptor-interacting protein serine/threonine kinase family, known to be involved in cell proliferation, differentiation and activation of transcription factors. Genetic variation within the locus is suggested to play a role in vulnerability to addictions. However, mechanism of action is still poorly understood. It has been suggested that may affect the development and/or functioning of dopaminergic pathways. To test this hypothesis, we generated a CRISPR-Cas9 loss of function zebrafish line causing a 27 bp insertion that disrupts the sequence introducing an early stop codon. We found that transcript levels were significantly lower in mutant ( ) fish compared to their wild type ( ) siblings. In adult zebrafish brain, ankk1 protein was detected in isocortex, hippocampus, basolateral amygdala, mesencephalon, and cerebellum, resembling the mammalian distribution pattern. In contrast, ankk1 protein was reduced in the brain of fish. Quantitative polymerase chain reaction analysis revealed an increase in expression of mRNA in at both larval and adult stages. In adult zebrafish brain, drd2 protein was detected in cerebral cortex, cerebellum, hippocampus, and caudate homolog regions, resembling the pattern in humans. In contrast, drd2 expression was reduced in cortical regions of being predominantly found in the hindbrain. No differences in the number of cell bodies or axonal projections detected by anti-tyrosine hydroxylase immunostaining on 3 days post fertilization (dpf) larvae were found. Behavioral analysis revealed altered sensitivity to effects of both amisulpride and apomorphine on locomotion and startle habituation, consistent with a broad loss of both pre and post synaptic receptors. mutants showed reduced sensitivity to the effect of the selective dopamine receptor antagonist amisulpride on locomotor responses to acoustic startle and were differentially sensitive to the effects of the non-selective dopamine agonist apomorphine on both locomotion and habituation. Taken together, our findings strengthen the hypothesis of a functional relationship between and , supporting a role for in the maintenance and/or functioning of dopaminergic pathways. Further work is needed to disentangle 's role at different developmental stages.
锚蛋白重复序列和激酶结构域包含蛋白1（ANKK1）是受体相互作用蛋白丝氨酸/苏氨酸激酶家族的成员，已知其参与细胞增殖、分化以及转录因子的激活。该基因座内的遗传变异被认为在成瘾易感性中起作用。然而，其作用机制仍知之甚少。有人提出，ANKK1可能会影响多巴胺能通路的发育和/或功能。为了验证这一假设，我们构建了一种CRISPR-Cas9功能缺失的斑马鱼品系，该品系导致27个碱基对的插入，破坏了ANKK1序列，引入了一个提前终止密码子。我们发现，与野生型（WT）同胞相比，ANKK1突变体（ankk1-/-）鱼的转录水平显著降低。在成年斑马鱼大脑中，在同皮质、海马体、基底外侧杏仁核、中脑和小脑中检测到ankk1蛋白，类似于哺乳动物的分布模式。相比之下，ankk1-/-鱼大脑中的ankk1蛋白减少。定量聚合酶链反应分析显示，在幼虫和成年阶段，ankk1-/-中多巴胺受体D2（drd2）mRNA的表达均增加。在成年斑马鱼大脑中，在大脑皮层、小脑、海马体和尾状核同源区域检测到drd2蛋白，类似于人类的模式。相比之下，drd2在ankk1-/-的皮质区域表达减少，主要在后脑中发现。在受精后3天（dpf）的幼虫中，通过抗酪氨酸羟化酶免疫染色未发现细胞体数量或轴突投射的差异。行为分析显示，对阿立哌唑和阿扑吗啡对运动和惊吓习惯化的影响的敏感性发生改变，这与突触前和突触后受体的广泛丧失一致。ankk1-/-突变体对选择性多巴胺受体拮抗剂阿立哌唑对听觉惊吓运动反应的影响敏感性降低，并且对非选择性多巴胺激动剂阿扑吗啡对运动和习惯化的影响具有不同的敏感性。综上所述，我们的研究结果强化了ANKK1与drd2之间存在功能关系的假设，支持ANKK1在多巴胺能通路的维持和/或功能中发挥作用。需要进一步的工作来阐明ANKK1在不同发育阶段的作用。
