Alsin/Rac1 signaling controls survival and growth of spinal motoneurons
Alsin/Rac1 signaling controls survival and growth of spinal motoneurons
复制标题
DOI：
10.1002/ana.20886
复制
发表时间：
2006-07-01
影响因子：
11.2
通讯作者：
Haase, Georg
中科院分区：
文献类型：
--
作者：
Jacquier, Arnaud;Buhler, Emmanuelle;Haase, Georg
目的：Alsin是GTP酶Rab 5和Rac 1的鸟嘌呤核苷酸交换因子，Alsin的隐性突变可导致幼年型肌萎缩侧索硬化症（ALS 2）和相关的运动神经元疾病。Alsin在运动神经元中的功能仍然不清楚，因为Alsin敲除小鼠不出现明显的运动神经元变性的迹象。方法：为了在ALS相关细胞类型中产生Alsin功能丧失模型，我们开发了一种新的小干扰RNA电穿孔技术，该技术可以有效地敲除胚胎大鼠脊髓运动神经元中的Alsin。小干扰RNA介导的alsin敲低后，培养的运动神经元显示EEA 1标记的早期内体的表观大小减少，转铁蛋白和LICAM的细胞内积累增加。Alsin基因敲低可诱导32%~ 48%的运动神经元死亡，并显著抑制存活神经元的轴突生长。这两种细胞表型的显性负Rac 1突变体的表达模仿，并完全封闭的组成型活性Rac 1突变体的表达。显性负或组成型活性形式的Rab 5的表达没有这样的effects.Interpretation：我们的数据表明，alsin控制运动神经元的生长和生存在Rac 1依赖的方式。这里报道的策略说明了小干扰RNA电穿孔如何用于产生涉及功能丧失机制的神经退行性疾病的细胞模型。
Objective: Recessive mutations in alsin, a guanine-nucleotide exchange factor for the GTPases Rab5 and Rac1, cause juvenile amyotrophic lateral sclerosis (ALS2) and related motoneuron disorders. Alsin function in motoneurons remained unclear because alsin knock-out mice do not develop overt signs of motoneuron degeneration.Methods: To generate an alsin loss-of-function model in an ALS-relevant cell type, we developed a new small interfering RNA electroporation technique that allows efficient knock down of alsin in embryonic rat spinal motoneurons.Results: After small interfering RNA-mediated alsin knockdown, cultured motoneurons displayed a reduced apparent size of EEA1-labeled early endosomes and an increased intracellular accumulation of transferrin and LICAM. Alsin knockdown induced cell death in 32 to 48% of motoneurons and significantly inhibited axon growth in the surviving neurons. Both cellular phenotypes were mimicked by expression of a dominant-negative Rac1 mutant and were completely blocked by expression of a constitutively active Rac1 mutant. Expression of dominant-negative or constitutively active forms of Rab5 had no such effects.Interpretation: Our data demonstrate that alsin controls the growth and survival of motoneurons in a Rac1-dependant manner. The strategy reported here illustrates how small interfering RNA electroporation can be used to generate cellular models of neurodegenerative disease involving a loss-of-function mechanism.