Hurelbrink C B, Armstrong R J, Luheshi L M, Dunnett S B, Rosser A E, Barker R A
Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge, CB2 2PY, UK.
Exp Neurol. 2001 Sep;171(1):46-58. doi: 10.1006/exnr.2001.7749.
Caspases are cysteine proteases involved in apoptotic cell death, and pharmacological caspase inhibition has been demonstrated to prevent neuronal cell death in certain experimental paradigms. In this study, the role of caspase-1 and -3 in the death of dopaminergic neurons derived from the E14 rat ventral mesencephalon (VM) has been examined in two model systems using peptide caspase inhibitors. First, cell death was induced in vitro by withdrawing serum after 2 days. Different doses of caspase-1 (IL-1beta converting enzyme) and caspase-3 inhibitors (Ac-DEVD-cmk) were added to the medium at the time of serum withdrawal, and the ability of the inhibitors to promote dopaminergic neuronal survival and prevent activation of caspase-3 was assessed at 7 days. Immunostaining using tyrosine hydroxylase (TH) and cleaved caspase-3 antibodies demonstrated that caspase-1 and -3 inhibitors reduce caspase-3 activation as well as overall cell death. This did not, however, improve the survival of TH-positive neurons, although it did appear to promote their maturation. The second paradigm investigated the effects of these inhibitors in the 6-hydroxydopamine rat model of PD, and similarly, addition of caspase-1 or -3 inhibitor during tissue preparation or immediately prior to grafting of VM tissue did not promote dopaminergic neuronal survival. These results demonstrate that the reduction of apoptotic cell death by pharmacological inhibition of caspase-1 and -3 does not increase dopaminergic neuronal survival in these paradigms and suggest either that caspase-3 activation is not the major determinant of dopaminergic neuronal death in vitro and in grafts or that the ability of caspase inhibitors to rescue cells depends upon the degree of apoptotic stress. This implies that strategies to improve dopaminergic cell survival in clinical programmes of transplantation for PD will need to target other pathways of cell death.
半胱天冬酶是参与凋亡性细胞死亡的半胱氨酸蛋白酶，在某些实验范式中，药理学上抑制半胱天冬酶已被证明可预防神经元细胞死亡。在本研究中，使用肽类半胱天冬酶抑制剂在两个模型系统中研究了半胱天冬酶-1和-3在源自E14大鼠腹侧中脑（VM）的多巴胺能神经元死亡中的作用。首先，在体外培养2天后通过去除血清诱导细胞死亡。在去除血清时向培养基中添加不同剂量的半胱天冬酶-1（白细胞介素-1β转换酶）和半胱天冬酶-3抑制剂（Ac-DEVD-cmk），并在7天时评估抑制剂促进多巴胺能神经元存活和预防半胱天冬酶-3激活的能力。使用酪氨酸羟化酶（TH）和裂解的半胱天冬酶-3抗体进行免疫染色表明，半胱天冬酶-1和-3抑制剂可减少半胱天冬酶-3的激活以及总体细胞死亡。然而，这并未提高TH阳性神经元的存活率，尽管它似乎确实促进了它们的成熟。第二个范式研究了这些抑制剂在帕金森病的6-羟基多巴胺大鼠模型中的作用，同样，在组织制备期间或即将移植VM组织之前添加半胱天冬酶-1或-3抑制剂并不能促进多巴胺能神经元的存活。这些结果表明，在这些范式中，通过药理学抑制半胱天冬酶-1和-3来减少凋亡性细胞死亡并不会增加多巴胺能神经元的存活，并表明要么半胱天冬酶-3激活不是体外和移植中多巴胺能神经元死亡的主要决定因素，要么半胱天冬酶抑制剂拯救细胞的能力取决于凋亡应激的程度。这意味着在帕金森病移植临床方案中改善多巴胺能细胞存活的策略将需要针对其他细胞死亡途径。
