Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia.
O'Brien Institute, 42 Fitzroy Street, Fitzroy, Victoria 3065, Australia; Centre for Eye Research Australia, University of Melbourne, Gisborne Street, East Melbourne 3002, Australia.
J Biol Chem. 2013 Apr 5;288(14):9696-9709. doi: 10.1074/jbc.M112.419713. Epub 2013 Feb 11.
Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.
谷氨酸受体过度刺激引起的兴奋毒性是脑缺血性中风神经元死亡的主要原因。过度刺激的离子型谷氨酸受体通过过度激活钙蛋白酶发挥其神经毒性作用，钙蛋白酶通过催化特定细胞蛋白的有限水解来诱导神经元死亡。在这里，我们报告说，在培养的皮质神经元中和在局灶性缺血性中风的大鼠模型中，Src 酪氨酸激酶被钙蛋白酶在 N 端独特结构域的一个位点切割。这产生了一个约 52 kDa 的截断 Src 片段，我们将其主要定位到细胞质中。一种源自 Src 独特结构域的细胞膜通透融合肽可防止钙蛋白酶在神经元中切割 Src，并防止兴奋毒性神经元死亡。为了探讨截断 Src 片段在神经元死亡中的作用，我们在培养的神经元中表达了重组的截断 Src 片段，并研究了它如何影响神经元的存活。表达这种片段，它缺乏豆蔻酰化基序和独特结构域，足以诱导神经元死亡。此外，生存促进激酶 Akt 的失活是其神经毒性信号通路中的关键步骤。由于 Src 维持神经元的存活，我们的结果表明钙蛋白酶切割是一种分子开关，将 Src 从促进细胞存活的分子转换为兴奋毒性中神经元死亡的介导者。除了揭示 Src 的新病理作用外，我们还发现截断 Src 片段的神经毒性作用表明了新的治疗策略，有可能最大限度地减少缺血性中风中的脑损伤。
