KBTBDN调控HnRNPA2/B1泛素化在常染色体隐性遗传小脑共济失调发病机制中的作用研究

KBTBDN was a novel causative gene of autosomal recessive cerebellar ataxias (ARCA) cloned through exome sequencing analysis in our previous work. We further created two mice models, KBTBDN knock out mice and KBTBDN P64L knock-in mice. Both mice models exhibit abnormal cerebellar development and impaired motor balance ability. KBTBDN can interact with CUL3 to form an E3 ubiquitin ligase and act as a substrate adaptor in this complex. Mass spectrometry analysis revealed that HnRNPA2/B1 is an interaction protein of KBTBDN, and HnRNPA2/B1 is a pathogenic gene of amyotrophic lateral sclerosis (ALS), which plays a key role in neurons. In summary, we hypothesized that the abnormal regulation of HTBRNDN2/B1 by KBTBDN may be an important pathological mechanism for ARCA. In the present study, our aim is to investigate the how KBTBDN regulates HnRNPA2/B1 ubiquitination modification and degradation, and how mutations in KBTBDN affect HnRNPA2/B1 stability and lead to neuron degeneration at cellular and animal level. In the mean while, we will try to rescue the KBTBDN KO/KI mice phenotype through regulation of HnRNPA2/B1 level. All these might help us to understand the pathological function of HnRNPA2/B1 in the development of KBTBDN-associated cerebellar ataxia, providing new clues for pathogenesis and potential therapeutic targets of ARCA.

本课题组前期工作克隆了新的常染色体隐性遗传小脑共济失调致病基因KBTBDN，该基因敲除/点突变小鼠具有小脑共济失调表型，功能研究示KBTBDN参与形成E3泛素连接酶复合体，调控底物蛋白降解。质谱分析发现HnRNPA2/B1是KBTBDN的互作蛋白，而HnRNPA2/B1是肌萎缩侧索硬化症(ALS)致病基因，对神经元意义重大。综上，我们提出假说KBTBDN对HnRNPA2/B1的调控异常可能是小脑共济失调发病的重要原因。本课题拟在此基础上，在细胞和动物水平研究KBTBDN调控HnRNPA2/B1泛素化修饰和降解的机制，及KBTBDN突变影响HnRNPA2/B1稳定性导致神经元变性的机制；通过调控HnRNPA2/B1水平，营救KBTBDN敲除/点突变小鼠表型，探讨HnRNPA2/B1在KBTBDN相关小脑共济失调发生中的重要作用，为揭示发病机制和潜在治疗靶点提供新的线索。

常染色体隐性小脑共济失调（ARCAs）是一组临床和遗传异质的罕见神经疾病。我们通过全外显子组测序（WES）和拷贝数变异（CNV）分析，在一隐性遗传的小脑共济失调家系中确定两个受影响的先证者在KBTBDN基因上携带纯合错义突变（c.191C>T，p.P64L）。KBTBDN是BTB结构域超家族的成员，它与CUL3相互作用，并作为泛素化过程中的底物衔接子。我们的研究表明，KBTBDN作为底物接头与基于CUL3的E3连接酶复合物相互作用，并介导HNRNPA2B1的泛素化和降解。重要的是，我们发现KBTBDN-P64L突变损害了KBTBDN蛋白的稳定性，并干扰了HNRNPA2B1的泛素化过程。在缺乏KBTBDN或携带KBTBDN P60L突变的小鼠中，HNRNPA2B1蛋白降解在体内受到显著抑制,并进一步影响小脑多种基因转录水平及可变剪接。与野生型小鼠相比，KBTBDN敲除小鼠和KBTBDN P60L/P60L敲除小鼠表现出运动平衡能力受损和小脑浦肯野细胞变性。这些结果表明KBTBDN是ARCA的一种新的致病基因，并为共济失调和RNP复合物之间的潜在机制联系提供了新的见解。