Kizil Caghan, Iltzsche Anne, Thomas Alvin Kuriakose, Bhattarai Prabesh, Zhang Yixin, Brand Michael
German Centre for Neurodegenerative Diseases (DZNE) Dresden within the Helmholtz Association, Arnoldstr. 18, 01307, Dresden, Germany; DFG-Center for Regenerative Therapies Dresden (CRTD) - Cluster of Excellence, Technische Universität Dresden, Fetscherstr. 105, 01307, Dresden, Germany.
DFG-Center for Regenerative Therapies Dresden (CRTD) - Cluster of Excellence, Technische Universität Dresden, Fetscherstr. 105, 01307, Dresden, Germany.
PLoS One. 2015 Apr 20;10(4):e0124073. doi: 10.1371/journal.pone.0124073. eCollection 2015.
Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.
斑马鱼的大脑能够在位于脑室区域的放射状胶质祖细胞（RGCs）的神经发生活动后再生丢失的神经元。了解这种能力背后的分子事件对于再生医学的转化研究具有极大的意义。因此，对RGCs和神经元中基因功能进行功能分析至关重要。通过脑室微量注射（CVMI），可以有效地靶向RGCs，但注射分子在脑组织较深部位（如含有神经元的实质区域）的穿透能力会显著降低。在本报告中，我们测试了五种已知的细胞穿透肽（CPPs）的穿透效率，并确定了两种——polyR和Trans——能够以剂量依赖的方式有效穿透脑组织且无明显毒性，这是通过TUNEL染色和L- plastin免疫组织化学确定的。我们还发现，经过一系列基于DBCO-PEG4-马来酰亚胺的迈克尔加成反应和叠氮介导的无铜点击反应的偶联反应后，polyR肽可以帮助携带质粒DNA进入脑组织达几个细胞直径的深度。结合CVMI的优点，如快速性、可重复性以及能够用于成年动物，CPPs提高了CVMI技术对中枢神经系统组织较深部位的适用性。
