Data_Sheet_1_Regional and age-dependent changes in ubiquitination in cellular and mouse models of spinocerebellar ataxia type 3.PDF

Spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease, is the most common dominantly inherited ataxia. SCA3 is caused by a CAG repeat expansion in the ATXN3 gene that encodes an expanded tract of polyglutamine in the disease protein ataxin-3 (ATXN3). As a deubiquitinating enzyme, ATXN3 regulates numerous cellular processes including proteasome- and autophagy-mediated protein degradation. In SCA3 disease brain, polyQ-expanded ATXN3 accumulates with other cellular constituents, including ubiquitin (Ub)-modified proteins, in select areas like the cerebellum and the brainstem, but whether pathogenic ATXN3 affects the abundance of ubiquitinated species is unknown. Here, in mouse and cellular models of SCA3, we investigated whether elimination of murine Atxn3 or expression of wild-type or polyQ-expanded human ATXN3 alters soluble levels of overall ubiquitination, as well as K48-linked (K48-Ub) and K63-linked (K63-Ub) chains. Levels of ubiquitination were assessed in the cerebellum and brainstem of 7- and 47-week-old Atxn3 knockout and SCA3 transgenic mice, and also in relevant mouse and human cell lines. In older mice, we observed that wild-type ATXN3 impacts the cerebellar levels of K48-Ub proteins. In contrast, pathogenic ATXN3 leads to decreased brainstem abundance of K48-Ub species in younger mice and changes in both cerebellar and brainstem K63-Ub levels in an age-dependent manner: younger SCA3 mice have higher levels of K63-Ub while older mice have lower levels of K63-Ub compared to controls. Human SCA3 neuronal progenitor cells also show a relative increase in K63-Ub proteins upon autophagy inhibition. We conclude that wild-type and mutant ATXN3 differentially impact K48-Ub- and K63-Ub-modified proteins in the brain in a region- and age-dependent manner.

脊髓小脑性共济失调3型（SCA3），亦称Machado-Joseph病，是最常见的显性遗传性共济失调。SCA3由编码疾病蛋白ataxin-3（ATXN3）中扩展的多聚谷氨酰胺的ATXN3基因中的CAG重复扩张引起。作为一种去泛素化酶，ATXN3调节包括蛋白酶体和自噬介导的蛋白质降解在内的多种细胞过程。在SCA3疾病大脑中，多聚Q扩展的ATXN3与其他细胞成分（如泛素化蛋白）一起在特定的区域（如小脑和脑干）积累，但致病性ATXN3是否影响泛素化物种的丰度尚不清楚。在本研究中，我们在SCA3小鼠和细胞模型中研究了消除小鼠Atxn3或表达野生型或多聚Q扩展的人ATXN3是否改变总体泛素化水平，以及K48连接（K48-Ub）和K63连接（K63-Ub）链的水平。在7周和47周大的Atxn3敲除和SCA3转基因小鼠的小脑和脑干中，以及相关的鼠和人类细胞系中评估了泛素化水平。在较老的小鼠中，我们观察到野生型ATXN3影响小脑中K48-Ub蛋白的水平。相反，致病性ATXN3导致较年轻小鼠脑干中K48-Ub物种的丰度降低，并且以年龄依赖的方式改变小脑和脑干中K63-Ub的水平：与对照相比，年轻的SCA3小鼠K63-Ub水平较高，而较老的小鼠K63-Ub水平较低。在自噬抑制后，人类SCA3神经元祖细胞也显示出K63-Ub蛋白的相对增加。我们得出结论，野生型和突变型ATXN3以区域和年龄依赖的方式不同地影响大脑中的K48-Ub和K63-Ub修饰的蛋白。