Moutal Aubin, François-Moutal Liberty, Brittain Joel M, Khanna May, Khanna Rajesh
Department of Pharmacology, College of Medicine, University of Arizona Tucson, AZ, USA.
Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine Indianapolis, IN, USA.
Front Cell Neurosci. 2015 Jan 26;8:471. doi: 10.3389/fncel.2014.00471. eCollection 2014.
The microtubule-associated axonal specification collapsin response mediator protein 2 (CRMP2) is a novel target for neuroprotection. A CRMP2 peptide (TAT-CBD3) conjugated to the HIV transactivator of transcription (TAT) protein's cationic cell penetrating peptide (CPP) motif protected neurons in the face of toxic levels of Ca(2+) influx leaked in via N-methyl-D-aspartate receptor (NMDAR) hyperactivation. Here we tested whether replacing the hydrophilic TAT motif with alternative cationic (nona-arginine (R9)), hydrophobic (membrane transport sequence (MTS) of k-fibroblast growth factor) or amphipathic (model amphipathic peptide (MAP)) CPPs could be superior to the neuroprotection bestowed by TAT-CBD3. In giant plasma membrane vesicles (GPMVs) derived from cortical neurons, the peptides translocated across plasma membranes with similar efficiencies. Cortical neurons, acutely treated with peptides prior to a toxic glutamate challenge, demonstrated enhanced efflux of R9-CBD3 compared to others. R9-CBD3 inhibited N-methyl-D-aspartate (NMDA)-evoked Ca(2+) influx to a similar extent as TAT-CBD3 while MTS-CBD3 was ineffective which correlated with the ability of R9- and TAT-CBD3, but not MTS-CBD3, to block NMDAR interaction with CRMP2. Unrestricted Ca(2+) influx through NMDARs leading to delayed calcium dysregulation and neuronal cell death was blocked by all peptides but MAP-CBD3. When applied acutely for 10 min, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective. In contrast, long-term (>24 h) treatment with MTS-CBD3 conferred neuroprotection where TAT-CBD3 failed. Neither peptide altered surface trafficking of NMDARs. Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3. Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.
微管相关的轴突特异性塌陷反应介导蛋白2（CRMP2）是神经保护的一个新靶点。一种与HIV转录反式激活因子（TAT）蛋白的阳离子细胞穿透肽（CPP）基序偶联的CRMP2肽（TAT-CBD3），在面对通过N-甲基-D-天冬氨酸受体（NMDAR）过度激活而泄漏的毒性水平的Ca(2+)内流时，能保护神经元。在这里，我们测试了用替代的阳离子（九聚精氨酸（R9））、疏水（成纤维细胞生长因子的膜转运序列（MTS））或两亲性（模型两亲性肽（MAP））CPP取代亲水性TAT基序是否可能优于TAT-CBD3所赋予的神经保护作用。在源自皮层神经元的巨大质膜囊泡（GPMV）中，这些肽以相似的效率跨质膜转运。在毒性谷氨酸刺激之前用肽急性处理的皮层神经元显示，与其他肽相比，R9-CBD3的流出增强。R9-CBD3抑制N-甲基-D-天冬氨酸（NMDA）诱发的Ca(2+)内流的程度与TAT-CBD3相似，而MTS-CBD3无效，这与R9-和TAT-CBD3但不是MTS-CBD3阻断NMDAR与CRMP2相互作用的能力相关。所有肽（除MAP-CBD3外）均能阻断通过NMDAR的不受限制的Ca(2+)内流，这种内流会导致延迟的钙失调和神经元细胞死亡。当急性应用10分钟时，R9-CBD3在神经保护方面比TAT-CBD3更有效，而MTS-和MAP-CBD3无效。相反，在TAT-CBD3无效的情况下，用MTS-CBD3进行长期（>24小时）治疗可赋予神经保护作用。两种肽均未改变NMDAR的表面转运。与TAT-CBD3相比，MTS-CBD3肽赋予的神经保护作用可能是由于其摄取增加以及流出减少。总体而言，我们的结果表明，改变CPP可以赋予CBD3货物不同的神经保护潜力。
