Gene expression profiling of shRNA transduced rat hippocampal neurons

Sirtuin 6 (SIRT6), a member of the sirtuin family, acts as nicotinamide adenine dinucleotide-dependent protein deacetylase, mono-adenosine diphosphate-ribosyltransferase, and fatty acid deacylase and plays critical roles in inflammation, aging, glycolysis, and DNA repair. Accumulating evidence has suggested that SIRT6 is involved in brain functions such as neuronal differentiation, neurogenesis, and learning and memory. However, the precise molecular roles of SIRT6 during neuronal circuit formation are not well understood. In this study, we attemped to elucidate the molecular roles of SIRT6 on neurite development using a primary culture of hippocampal neurons. We observed that SIRT6 was abundantly localized in the nucleus, and its expression was markedly increased during neurite outgrowth and synaptogenesis. Using shRNA-mediated SIRT6 knockdown, we showed that the dendritic length and number of dendrite branches were significantly reduced in SIRT6 knockdown neurons. Microarray and subsequent gene ontology analysis revealed that reducing SIRT6 caused the downregulation of immediate early genes (IEGs) and alteration of several biological processes including MAPK (ERK1/2) signaling. We found that nuclear accumulation of phosphorylated ERK1/2 was significantly reduced in SIRT6 knockdown neurons. Overexpression of SIRT6 promoted dendritic length and branching, but the mutants lacking deacetylase activity had no significant effect on the dendritic morphology. Collectively, the presented findings reveal a role of SIRT6 in dendrite morphogenesis and suggest that SIRT6 may act as an important regulator of the ERK1/2 signaling pathway that mediates IEG expression, which leads to dendritic development.沉默信息调节因子6（Sirtuin 6, SIRT6）是沉默信息调节因子家族的一员，可作为烟酰胺腺嘌呤二核苷酸依赖型蛋白质去乙酰化酶、单腺苷二磷酸核糖基转移酶及脂肪酸去酰基酶发挥功能，在炎症反应、衰老、糖酵解与DNA修复过程中发挥关键调控作用。越来越多的研究证据表明，SIRT6参与神经元分化、神经发生以及学习记忆等脑功能调控。然而，目前人们对SIRT6在神经元环路形成过程中的确切分子机制仍不甚明晰。本研究尝试通过海马神经元原代培养体系，阐明SIRT6在神经元突起发育中的分子功能。研究观察到，SIRT6主要定位于细胞核内，且其表达在神经元突起生长与突触发生阶段显著上调。通过短发夹RNA（short hairpin RNA, shRNA）介导的SIRT6基因敲低实验，我们发现敲低SIRT6的神经元其树突长度与树突分支数量均显著降低。基因芯片（Microarray）分析与后续基因本体（Gene Ontology, GO）分析显示，抑制SIRT6的表达会导致即刻早期基因（immediate early genes, IEGs）的下调，并改变包括丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK，即ERK1/2）信号通路在内的多种生物学过程。我们进一步发现，在SIRT6敲低的神经元中，磷酸化ERK1/2的细胞核聚集现象显著减少。过表达SIRT6可促进神经元树突的长度与分支形成，而缺失去乙酰化酶活性的SIRT6突变体则对树突形态无显著影响。综上，本研究结果揭示了SIRT6在神经元树突形态发生中的作用，并提示SIRT6可能作为调控ERK1/2信号通路的重要调节因子，通过介导即刻早期基因的表达进而影响神经元树突发育。