Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish
Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish
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DOI：
10.1111/apha.13725
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发表时间：
2021-08-26
影响因子：
6.3
通讯作者：
Panula, Pertti
中科院分区：
文献类型：
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作者：
Baronio, Diego;Chen, Yu-Chia;Panula, Pertti
我们旨在利用一种新的斑马鱼模型，确定囊泡单胺转运蛋白2，也称为溶质载体蛋白18a2 (SLC18A2)（以下简称Vmat2）在脑单胺调节、它们的转换、行为和大脑发育中的潜在作用。方法利用CRISPR/Cas9系统构建缺乏功能性Vmat2的斑马鱼品系。监测幼虫行为和心率。用高压液相色谱法分析单胺及其代谢物。用定量PCR、原位杂交和免疫细胞化学分析了脑发育所必需的胺合成和降解酶以及基因。结果第3外显子5bp缺失引起早期移码，并在受精后2周内致死性。纯合突变体（以下简称突变体）在夜间表现出正常的低运动活动，但对光照变化的反应异常。在突变体中，多巴胺、去甲肾上腺素、5-羟色胺和组胺水平降低，而多巴胺和5-羟色胺代谢物水平升高，这意味着单胺含量升高。同样，组胺、5-羟色胺和多巴胺免疫反应细胞较少。细胞多巴胺免疫染色在突变体中缺失，在野生型幼虫中表达酪氨酸羟化酶1 （Th1）的神经元比表达th2的神经元更突出。尽管多巴胺水平降低，但突变体表现出多巴胺合成酶的上调。此外，在突变体中，表达组氨酸脱羧酶的神经元数量增加，notch1a和pax2a在脑增殖区下调。结论缺乏Vmat2会增加单胺的周转，并上调编码胺合成酶的基因，包括组氨酸脱羧酶。与干细胞发育有关的基因Notch1a和pax2a在突变体中下调。斑马鱼vmat2突变株可能是研究单胺转运如何影响大脑发育和功能以及用于药物筛选的有用模型。
Aim We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. Methods A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. Results The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2 weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. Conclusion Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.