Tousley Adelaide, Iuliano Maria, Weisman Elizabeth, Sapp Ellen, Zhang Ningzhe, Vodicka Petr, Alexander Jonathan, Aviolat Hubert, Gatune Leah, Reeves Patrick, Li Xueyi, Khvorova Anastasia, Ellerby Lisa M, Aronin Neil, DiFiglia Marian, Kegel-Gleason Kimberly B
Department of Neurology, Laboratory of Cellular Neurobiology, Massachusetts General Hospital, Charlestown, MA, USA.
Buck Institute for Research on Aging, Novato, CA, USA.
J Huntingtons Dis. 2019;8(1):53-69. doi: 10.3233/JHD-180311.
Previous studies suggest that Huntingtin, the protein mutated in Huntington's disease (HD), is required for actin based changes in cell morphology, and undergoes stimulus induced targeting to plasma membranes where it interacts with phospholipids involved in cell signaling. The small GTPase Rac1 is a downstream target of growth factor stimulation and PI 3-kinase activity and is critical for actin dependent membrane remodeling.
To determine if Rac1 activity is impaired in HD or regulated by normal Huntingtin.
Analyses were performed in differentiated control and HD human stem cells and HD Q140/Q140 knock-in mice. Biochemical methods included SDS-PAGE, western blot, immunoprecipitation, affinity chromatography, and ELISA based Rac activity assays.
Basal Rac1 activity increased following depletion of Huntingtin with Huntingtin specific siRNA in human primary fibroblasts and in human control neuron cultures. Human cells (fibroblasts, neural stem cells, and neurons) with the HD mutation failed to increase Rac1 activity in response to growth factors. Rac1 activity levels were elevated in striatum of 1.5-month-old HD Q140/Q140 mice and in primary embryonic cortical neurons from HD mice. Affinity chromatography analysis of striatal lysates showed that Huntingtin is in a complex with Rac1, p85α subunit of PI 3-kinase, and the actin bundling protein α-actinin and interacts preferentially with the GTP bound form of Rac1. The HD mutation reduced Huntingtin interaction with p85α.
These findings suggest that Huntingtin regulates Rac1 activity as part of a coordinated response to growth factor signaling and this function is impaired early in HD.
先前的研究表明，亨廷顿蛋白（在亨廷顿舞蹈病（HD）中发生突变的蛋白质）是基于肌动蛋白的细胞形态变化所必需的，并且会在刺激诱导下靶向质膜，在那里它与参与细胞信号传导的磷脂相互作用。小GTP酶Rac1是生长因子刺激和PI 3激酶活性的下游靶点，对肌动蛋白依赖性膜重塑至关重要。
确定Rac1活性在HD中是否受损或受正常亨廷顿蛋白调控。
在分化的对照和HD人类干细胞以及HD Q140/Q140基因敲入小鼠中进行分析。生化方法包括SDS-PAGE、蛋白质印迹、免疫沉淀、亲和层析和基于ELISA的Rac活性测定。
在人原代成纤维细胞和人对照神经元培养物中，用亨廷顿蛋白特异性siRNA耗尽亨廷顿蛋白后，基础Rac1活性增加。携带HD突变的人类细胞（成纤维细胞、神经干细胞和神经元）对生长因子无反应，无法增加Rac1活性。1.5月龄HD Q140/Q140小鼠的纹状体以及HD小鼠的原代胚胎皮质神经元中，Rac1活性水平升高。对纹状体裂解物的亲和层析分析表明，亨廷顿蛋白与Rac1、PI 3激酶的p85α亚基以及肌动蛋白成束蛋白α辅肌动蛋白形成复合物，并优先与Rac1的GTP结合形式相互作用。HD突变减少了亨廷顿蛋白与p85α的相互作用。
这些发现表明，亨廷顿蛋白作为对生长因子信号协同反应的一部分来调节Rac1活性，并且该功能在HD早期受损。
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