Data from: Extracellular matrix-associated gene expression in adult sensory neurons cultured on laminin substrates

Background: In our previous investigations of the role of the extracellular matrix (ECM) in promoting neurite growth we have observed that a permissive laminin (LN) substrate stimulates differential growth responses in subpopulations of mature dorsal root ganglion (DRG) neurons. DRG neurons expressing Trk and p75 receptors grow neurites on a LN substrate in the absence of neurotrophins, while isolectin B4-binding neurons (IB4+) do not display significant growth under the same conditions. We set out to determine whether there was an expression signature of the LN-induced neurite growth phenotype. Using a lectin binding protocol IB4+ neurons were isolated from dissociated DRG neurons, creating two groups - IB4+ and IB4-. A small-scale microarray approach was employed to screen the expression of a panel of ECM-associated genes following dissociation (t=0) and after 24 hr culture on LN (t=24LN). This was followed by qRT-PCR and immunocytochemistry of selected genes. Results: The microarray screen showed that 36 of the 144 genes on the arrays were consistently expressed by the neurons. The array analyses showed that six genes had lower expression in the IB4+ neurons compared to the IB4- cells at t=0 (CTSH, Icam1, Itgβ1, Lamb1, Plat, Spp1), and one gene was expressed at higher levels in the IB4+ cells (Plaur). qRT-PCR was carried out as an independent assessment of the array results. There were discrepancies between the two methods, with qRT-PCR confirming the differences in Lamb1, Plat and Plaur, and showing decreased expression of AdamTs1, FN, and Icam in the IB4+ cells at t=0. After 24 hr culture on LN, there were no significant differences detected by qRT-PCR between the IB4+ and IB4- cells. However, both groups showed upregulation of Itgβ1 and Plaur after 24 hr on LN, the IB4+ group also had increased Plat, and the IB4- cells showed decreased Lamb1, Icam1 and AdamTs1. Further, the array screen also detected a number of genes (not subjected to qRT-PCR) expressed similarly by both populations in relatively high levels but not detectably influenced by time in culture (Bsg, Cst3, Ctsb, Ctsd, Ctsl, Mmp14, Mmp19, Sparc. We carried out immunohistochemistry to confirm expression of proteins encoded by a number of these genes. Conclusions:Our results show that 1B4+ and IB4- neurons differ in the expression of several genes that are associated with responsiveness to the ECM prior to culturing (AdamTs1, FN, Icam1, Lamb1, Plat, Plaur). The data suggest that the genes expressed at higher levels in the IB4- neurons could contribute to the initial growth response of these cells in a permissive environment and could also represent a common injury response that subsequently promotes axon regeneration. The differential expression of several extracellular matrix molecules (FN, Lamb1, Icam) may suggest that the IB4- neurons are capable of maintaining /secreting their local extracellular environment which could aid in the regenerative process. Overall, these data provide new information on potential targets that could be manipulated to enhance axonal regeneration in the mature nervous system.研究背景：在我们此前关于细胞外基质（extracellular matrix, ECM）促进神经突生长的相关研究中，观察到允许性层粘连蛋白（laminin, LN）底物可诱导成熟背根神经节（dorsal root ganglion, DRG）神经元的不同亚群产生差异化生长反应。表达Trk与p75受体的DRG神经元可在无神经营养因子（neurotrophins）的条件下于LN底物上长出神经突，而异凝集素B4结合神经元（isolectin B4-binding neurons, IB4+）在相同培养条件下则无显著生长现象。本研究旨在明确LN诱导的神经突生长表型是否存在特异性表达特征。我们采用凝集素结合方案，从解离的DRG神经元中分离出IB4+神经元，由此得到两组样本——IB4+组与IB4-组。本研究采用小型基因芯片（microarray）技术，对解离后（t=0）以及在LN底物上培养24小时后（t=24LN）的一组细胞外基质相关基因的表达情况进行筛选；随后通过实时定量聚合酶链反应（qRT-PCR）与免疫细胞化学（immunocytochemistry）对部分候选基因进行验证。 研究结果：基因芯片筛选显示，芯片搭载的144个基因中，有36个可在神经元中稳定表达。芯片分析表明，在t=0时刻，相较于IB4-细胞，IB4+神经元中有6个基因表达量更低（CTSH、Icam1、Itgβ1、Lamb1、Plat、Spp1），另有1个基因在IB4+细胞中表达量更高（Plaur）。我们采用qRT-PCR作为独立验证方法以评估芯片结果，但两种方法存在一定差异：qRT-PCR证实了Lamb1、Plat与Plaur的表达差异，同时发现在t=0时刻，IB4+细胞中AdamTs1、FN与Icam的表达量更低。在LN底物上培养24小时后，qRT-PCR未检测到IB4+与IB4-细胞间存在显著表达差异；不过两组样本在LN上培养24小时后均出现Itgβ1与Plaur的表达上调，其中IB4+组还出现Plat表达升高，而IB4-组则出现Lamb1、Icam1与AdamTs1的表达下调。此外，基因芯片筛选还检测到多个基因（未进行qRT-PCR验证）在两组神经元中表达水平相似且均较高，但不受培养时间的影响（Bsg、Cst3、Ctsb、Ctsd、Ctsl、Mmp14、Mmp19、Sparc）。我们通过免疫组织化学（immunohistochemistry）验证了部分上述基因编码蛋白的表达情况。 研究结论：本研究结果显示，在培养前，IB4+与IB4-神经元在多个与细胞外基质响应相关的基因表达上存在差异（AdamTs1、FN、Icam1、Lamb1、Plat、Plaur）。数据表明，在IB4-神经元中高表达的基因可能有助于这些细胞在允许性环境中的初始生长反应，同时也可能代表一种后续可促进轴突再生的常见损伤应答机制。多种细胞外基质分子（FN、Lamb1、Icam）的差异化表达提示，IB4-神经元能够维持或分泌其局部细胞外基质环境，这或许可辅助再生过程。总体而言，本研究为可用于调控成熟神经系统轴突再生的潜在靶点提供了新的科学依据。