We have previously demonstrated that Cationic Arginine-Rich Peptides (CARPs) and in particular poly-arginine-18 (R18; 18-mer of arginine) exhibit potent neuroprotective properties in both in vitro and in vivo neuronal injury models. Based on the current literature, there is a consensus that arginine residues by virtue of their positive charge and guanidinium head group is the critical element for imparting CARP neuroprotective properties and their ability to traverse cell membranes. This study examined the importance of guanidinium head groups in R18 for peptide cellular uptake, localization, and neuroprotection. This was achieved by using poly-ornithine-18 (O18; 18-mer of ornithine) as a control, which is structurally identical to R18, but possesses amino head groups rather than guanidino head groups. Epifluorescence and confocal fluorescence microscopy was used to examine the cellular uptake and localization of the FITC-conjugated R18 and O18 in primary rat cortical neurons and SH-SY5Y human neuroblastoma cell cultures. An in vitro cortical neuronal glutamic acid excitotoxicity model was used to compare the effectiveness of R18 and O18 to inhibit cell death and intracellular calcium influx, as well as caspase and calpain activation. Fluorescence imaging studies revealed cellular uptake of both FITC-R18 and FITC-O18 in neuronal and SH-SY5Y cells; however, intracellular localization of the peptides differed in neurons. Following glutamic acid excitotoxicity, only R18 was neuroprotective, prevented caspases and calpain activation, and was more effective at reducing neuronal intracellular calcium influx. Overall, this study demonstrated that for long chain cationic poly-arginine peptides, the guanidinium head groups provided by arginine residues are an essential requirement for neuroprotection but are not required for entry into neurons.

中文翻译：

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聚精氨酸18（R18）和聚鸟氨酸18（O18）肽的体外细胞吸收和神经保护功效：精氨酸胍基头部基团对神经保护的关键作用。

我们以前已经证明，富含阳离子精氨酸的肽（CARPs），尤其是聚精氨酸18（R18；精氨酸的18-mer）在体外和体内神经元损伤模型中均显示出强大的神经保护特性。根据目前的文献，已经达成共识，精氨酸残基凭借其正电荷和胍基头部基团是赋予CARP神经保护特性及其穿越细胞膜能力的关键元素。这项研究检查了R18中胍基头基对于肽细胞摄取，定位和神经保护的重要性。这是通过使用聚鸟氨酸-18（O18；鸟氨酸的18-mer）作为对照来实现的，其在结构上与R18相同，但具有氨基头部基团而不是胍基头部基团。使用落射荧光和共聚焦荧光显微镜检查了FITC偶联的R18和O18在原代大鼠皮层神经元和SH-SY5Y人成神经细胞瘤细胞培养物中的细胞摄取和定位。体外皮层神经元谷氨酸兴奋性毒性模型用于比较R18和O18抑制细胞死亡和细胞内钙内流以及胱天蛋白酶和钙蛋白酶激活的有效性。荧光成像研究揭示了神经元和SH-SY5Y细胞中FITC-R18和FITC-O18的细胞摄取。然而，肽在细胞内的定位在神经元中是不同的。谷氨酸兴奋性中毒后，仅R18具有神经保护作用，可防止胱天蛋白酶和钙蛋白酶活化，并且在减少神经元细胞内钙内流方面更为有效。全面的，