Wang Kevin K W, Yang Zhihui, Chiu Allen, Lin Fan, Rubenstein Richard
Program for Neurotrauma, Neuroproteomics and Biomarkers Research, Departments of Psychiatry, Neuroscience and Physiological Science, McKnight Brain Institute, University of Florida, 1149 South Newell Drive, Gainesville, FL, 32611, USA.
Laboratory of Neurodegenerative Diseases and CNS Biomarker Discovery, Departments of Neurology and Physiology/Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Avenue, Box #1213, Brooklyn, NY, 11203-2098, USA.
Mol Neurobiol. 2016 Sep;53(7):4821-32. doi: 10.1007/s12035-015-9407-8. Epub 2015 Sep 4.
Overexpression of cellular prion protein, PrP(C), has cytoprotective effects against neuronal injuries. Inhibition of cell death-associated proteases such as necrosis-linked calpain and apoptosis-linked caspase are also neuroprotective. Here, we systematically studied how PrP(C) expression levels and cell death protease inhibition affect cytotoxic challenges to both neuronal and glial cells in mouse cerebrocortical mixed cultures (CCM). Primary CCM derived from three mouse lines expressing no (PrP(C) knockout mice (PrPKO)), normal (wild-type (wt)), or high (tga20) levels of PrP(C) were subjected to necrotic challenge (calcium ionophore A23187) and apoptotic challenge (staurosporine (STS)). CCM which originated from tga20 mice provided the most robust neuron-astroglia protective effects against necrotic and early apoptotic cell death (lactate dehydrogenase (LDH) release) at 6 h but subsequently lost its cytoprotective effects. In contrast, PrPKO-derived cultures displayed elevated A23187- and STS-induced cell death at 24 h. Calpain inhibitor SNJ-1945 protected against A23187 challenge at 6 h in CCM from all three mouse lines but protected only against A23187 and STS treatments by 24 h in the PrPKO line. In parallel, caspase inhibitor Z-D-DCB protected against pro-apoptotic STS challenge at 6 and 24 h. Furthermore, we also examined αII-spectrin breakdown products (primarily from neurons) and glial fibrillary acidic protein (GFAP) breakdown products (from astroglia) as cytoskeletal proteolytic biomarkers. Overall, it appeared that both neurons and astroglial cells were less vulnerable to proteolytic attack during A23187 and STS challenges in tga20-derived cultures but more vulnerable in PrPKO-derived cultures. In addition, calpain and caspase inhibitors provide further protection against respective protease attacks on these neuronal and glial cytoskeletal proteins in CCM regardless of mouse-line origin. Lastly, some synergistic cytoprotective effects between PrP(C) expression and addition of cell death-linked protease inhibitors were also observed.
细胞朊蛋白PrP(C)的过表达对神经元损伤具有细胞保护作用。抑制与细胞死亡相关的蛋白酶，如与坏死相关的钙蛋白酶和与凋亡相关的半胱天冬酶，也具有神经保护作用。在此，我们系统地研究了PrP(C)表达水平和细胞死亡蛋白酶抑制如何影响小鼠大脑皮质混合培养物(CCM)中神经元和神经胶质细胞的细胞毒性挑战。从三种表达水平不同的小鼠品系获得的原代CCM，分别是不表达PrP(C)的小鼠(PrP(C)基因敲除小鼠(PrPKO))、正常表达水平的小鼠(野生型(wt))以及高表达水平的小鼠(tga20)，对其进行坏死性挑战(钙离子载体A23187)和凋亡性挑战(星形孢菌素(STS))。源自tga20小鼠的CCM在6小时时对坏死性和早期凋亡性细胞死亡(乳酸脱氢酶(LDH)释放)提供了最强的神经元-星形胶质细胞保护作用，但随后失去了其细胞保护作用。相比之下，源自PrPKO的培养物在24小时时显示出A23187和STS诱导的细胞死亡增加。钙蛋白酶抑制剂SNJ-1945在6小时时对来自所有三种小鼠品系的CCM中的A23187挑战具有保护作用，但在24小时时仅对PrPKO品系中的A23187和STS处理具有保护作用。同时，半胱天冬酶抑制剂Z-D-DCB在6小时和24小时时对促凋亡的STS挑战具有保护作用。此外，我们还检测了αII-血影蛋白降解产物(主要来自神经元)和胶质纤维酸性蛋白(GFAP)降解产物(来自星形胶质细胞)作为细胞骨架蛋白水解生物标志物。总体而言，在源自tga20的培养物中，在A23187和STS挑战期间，神经元和星形胶质细胞似乎对蛋白水解攻击的敏感性较低，而在源自PrPKO的培养物中则更敏感。此外，无论小鼠品系来源如何，钙蛋白酶和半胱天冬酶抑制剂都能进一步保护CCM中的这些神经元和神经胶质细胞骨架蛋白免受各自蛋白酶的攻击。最后，还观察到PrP(C)表达与添加细胞死亡相关蛋白酶抑制剂之间存在一些协同细胞保护作用。
