Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité-Universitätsmedizin Berlin, Berlin, Germany.
BMC Neurosci. 2013 Mar 19;14:36. doi: 10.1186/1471-2202-14-36.
Recently, we and others proposed plasticity-related gene 3 (PRG3) as a novel molecule in neuritogenesis based on PRG3 overexpression experiments in neuronal and non-neuronal cell lines. However, direct information on PRG3 effects in neuronal development and, in particular, its putative spatio-temporal distribution and conditions of action, is sparse.
We demonstrate here that PRG3 induces filopodia formation in HEK293 cells depending on its N-glycosylation status. The PRG3 protein was strongly expressed during mouse brain development in vivo from embryonic day 16 to postnatal day 5 (E16 - P5). From P5 on, expression declined. Furthermore, in early, not yet polarized hippocampal cultured neurons, PRG3 was expressed along the neurite shaft. Knock-down of PRG3 in these neurons led to a decreased number of neurites. This phenotype is rescued by expression of an shRNA-resistant PRG3 construct in PRG3 knock-down neurons. After polarization, endogenous PRG3 expression shifted mainly to axons, specifically to the plasma membrane along the neurite shaft. These PRG3 pattern changes appeared temporally and spatially related to ongoing synaptogenesis. Therefore we tested (i) whether dendritic PRG3 re-enhancement influences synaptic currents and (ii) whether synaptic inputs contribute to the PRG3 shift. Our results rendered both scenarios unlikely: (i) PRG3 over-expression had no influence on miniature excitatory postsynaptic currents (mEPSC) and (ii) blocking of incoming signals did not alter PRG3 distribution dynamics. In addition, PRG3 levels did not interfere with intrinsic neuronal properties.
Taken together, our data indicate that endogenous PRG3 promotes neurite shaft protrusion and therefore contributes to regulating filopodia formation in immature neurons. PRG3 expression in more mature neurons, however, is predominantly localized in the axon. Changes in PRG3 levels did not influence intrinsic or synaptic neuronal properties.
最近，我们和其他人基于在神经元和非神经元细胞系中过表达 PRG3 的实验，提出了 PRG3（多结构域蛋白 3）是神经突发生过程中的一种新型分子。然而，关于 PRG3 对神经元发育的直接影响，特别是其潜在的时空分布和作用条件的信息还很稀少。
我们在这里证明 PRG3 依赖其 N-糖基化状态诱导 HEK293 细胞形成丝状伪足。PRG3 蛋白在体内从胚胎 16 天到出生后 5 天（E16-P5）在小鼠大脑发育过程中强烈表达。从 P5 开始，表达量下降。此外，在早期、尚未极化的海马培养神经元中，PRG3 沿轴突表达。在这些神经元中敲低 PRG3 会导致轴突数量减少。这种表型可以通过在 PRG3 敲低神经元中表达 shRNA 抗性 PRG3 构建体来挽救。极化后，内源性 PRG3 表达主要转移到轴突，特别是沿着轴突的质膜。这些 PRG3 模式变化在时间和空间上与正在进行的突触发生有关。因此，我们测试了（i）树突状 PRG3 的再增强是否影响突触电流，以及（ii）突触输入是否有助于 PRG3 的转移。我们的结果表明这两种情况都不太可能：（i）PRG3 的过表达对微小兴奋性突触后电流（mEPSC）没有影响，（ii）阻断传入信号不会改变 PRG3 分布动力学。此外，PRG3 水平不会干扰内在神经元特性。
综上所述，我们的数据表明，内源性 PRG3 促进轴突干突出，从而有助于调节未成熟神经元中的丝状伪足形成。然而，在更成熟的神经元中，PRG3 的表达主要定位于轴突。PRG3 水平的变化不会影响内在或突触神经元特性。
