Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin
Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin
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DOI：
10.1007/bf03401784
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发表时间：
2000-05-01
影响因子：
5.7
通讯作者：
Andrade-Gordon, P
中科院分区：
文献类型：
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作者：
Festoff, BM;D'Andrea, MR;Andrade-Gordon, P
背景：人们正在积极寻找神经退行性变的潜在机制以寻求新的治疗策略。转基因、基因敲除和遗传小鼠模型极大地帮助我们了解神经元细胞死亡的机制。一种自然发生的常染色体隐性突变体（称为 wobbler）和家族性肌萎缩侧索硬化症 (FALS(1)) 超氧化物歧化酶 (SOD)1 突变的转基因小鼠已经存在，但其分子机制仍然未知。两种表型在出生后均可检测到。摇摆在生命的第三周就可以检测到，此时纯合子（wr/wr）在颈椎中表现出明显的神经胶质增生和显着的运动神经元损失，但在腰椎和脊髓节段中却没有。为了解决分子机制，我们评估了与多功能丝氨酸蛋白酶凝血酶相关的“死亡信号”，凝血酶通过裂解 G 蛋白偶联的蛋白酶激活受体 1 (PAR-1) 导致培养物中的运动神经元细胞凋亡。材料和方法：通过显色底物测定测定凝血酶活性，使用抗 PAR-1 进行蛋白质免疫印迹和免疫组织化学，以观察受体和受体的定位。抗GFAP染色用于监测星形细胞增多症。通过逆转录聚合酶链式反应 (qRT-PCR) 和原位杂交测定 PAR-1 mRNA 水平和位置。通过原位 DNA 片段化分析监测细胞死亡。结果：在初步研究中，我们发现 wr/wr 颈脊髓中 PAR-1 mRNA 与野生型 (wt) 同窝小鼠相比增加了 5 倍。我们目前的研究表明，反应性星形细胞增多和运动神经元细胞死亡与凝血酶信号传导的改变存在因果关系。免疫细胞化学证明并通过原位杂交证实，与野生型相比，表型 wr/wr 运动神经元中 PAR-1 蛋白表达增加，但星形胶质细胞中没有。与腰椎相比，颈椎的这种增加要大得多，与运动神经元变性平行。我们还发现，使用来自基因分型胚胎的 RNA 的逆转录聚合酶链反应 (qRT-PCR)，在 E12 的 wr/wr 脐带中 PAR-1 已经增加，这是最早检查的时间。 结论：因此，在 wobbler 小鼠中，运动神经元变性和死亡在时间上和地形上都遵循 PAR-1 的表达。由于我们的培养研究表明凝血酶通过激活 PAR-1 动员 [Ca2+](i)，最终导致运动神经元凋亡，因此发育过程中 PAR-1 的上调可能会导致 wobbler 中“适当”和“不适当”的神经元死亡。
Background: Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS(1)) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated "death signals" associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1(PAR-1).Materials and Methods: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridizations. Cell death was monitored with in situ DNA fragmentation assays.Results: In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) littermates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wr motor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar, segments, paralleling motor neuron degeneration. We also found, using reverse transcription polymerase chain reaction (qRT-PCR) with RNA from genotyped embryos, that PAR-1 was already increased in wr/wr cords at E12, the earliest time examined.Conclusions: Thus, motor neuron degeneration and death follows PAR-1 expression both temporally and topographically in wobbler mice. Since our culture studies show that thrombin mobilized [Ca2+](i) by activating PAR-1, eventually leading to motor neuron apoptosis, up-regulation of PAR-1 during development may contribute both to "appropriate" as well as "inappropriate" neuronal death in wobbler.