The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD

Expanded intronic G4C2 repeats in the C9ORF72 gene cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These intronic repeats are translated through a non-AUG-dependent mechanism into five different dipeptide repeat proteins (DPRs), including poly-glycine-arginine (GR), which is aggregation-prone and neurotoxic. Here, we report that Kapβ2 and GR interact, co-aggregating, in cultured neurons in vitro and CNS tissue in vivo. Importantly, this interaction significantly decreased the risk of death of cultured GR-expressing neurons. Downregulation of Kapβ2 is detrimental to their survival, whereas increased Kapβ2 levels mitigated GR-mediated neurotoxicity. As expected, GR-expressing neurons displayed TDP-43 nuclear loss. Raising Kapβ2 levels did not restore TDP-43 into the nucleus, nor did alter the dynamic properties of GR aggregates. Overall, our findings support the design of therapeutic strategies aimed at up-regulating Kapβ2 expression levels as a potential new ..., Human tissues: Human post-mortem CNS tissue from healthy controls and patients carrying the C9ORF72 intronic nucleotide repeat expansion was obtained from the Target ALS and the Jefferson Weinberg ALS center biobanks. Information and demographics of patients and controls are presented in Table 4. Human post-mortem tissues (fresh frozen) were stored at -80 C. Cell cultures: HEK293 cells were cultured in DMEM medium (Cytiva Cat.# SH30243.LS ) supplemented with 10% FBS (Cytiva Cat.# SH30071.01HI), penicillin, and streptomycin (Thermo Fisher Scientific Cat.# SV30010). Cells were passaged every 3-4 days using 0.05% trypsin (Corning Cat.# 25-051-C). Primary cortical neurons: After meninges removal, brains were dissected from embryonic day 16 (E16) rat embryos. Cortices and midbrain regions were cut into small pieces and incubated on a shaker at 80 rcf for 45 min at 37°C in 0.2% trypsin in HBSS without Ca2+ and Mg2+ (Cytiva Cat.# SH30588.01). FBS (Cytiva Cat.# SH30071.01HI) was added, and the ..., , # The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD [https://doi.org/10.5061/dryad.v41ns1s4f](https://doi.org/10.5061/dryad.v41ns1s4f) These datasets contain all the source files for the graphs in the associated paper. ## Description of the data and file structure Source data for each graph labeled as panel yx (y=number of figure, x=letter of the panel) ### Figure 1. GR does not affect Kapβ2 levels in neurons. * a) qPCR analysis of Kapβ2 levels in cortical neurons transduced with GFP or GR50. Data are represented as mean ± S.E.M. (n=6 biological replicates, Student t-test, n.s. = not significant) * c) The graph bar shows the quantification of the WB of cortical neuron extracts for Kapβ2 protein. Total protein staining was used to normalize. Data are represented as mean ± S.E.M. (n=3 biological replicates, Student t-test, n.s. = not significant). * d) qPCR analysis of Kapβ2 transcript levels in the cortex and spinal cord o...C9ORF72基因中扩增的内含子G4C2重复序列可引发肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）与额颞叶痴呆（frontotemporal dementia, FTD）。此类内含子重复序列可通过非AUG依赖的翻译机制，生成五种不同的二肽重复蛋白（dipeptide repeat proteins, DPRs），其中包括多聚甘氨酸-精氨酸（poly-glycine-arginine, GR）——该蛋白易发生聚集且具有神经毒性。本研究证实，核输入受体Kapβ2与GR可发生相互作用并共同聚集，该现象在体外培养神经元以及体内中枢神经系统（central nervous system, CNS）组织中均存在。值得注意的是，该相互作用可显著降低表达GR的培养神经元的死亡风险。下调Kapβ2的表达会损害神经元存活，而提升Kapβ2的水平则可缓解GR介导的神经毒性。如预期所示，表达GR的神经元出现了TDP-43的核缺失现象。提升Kapβ2的水平既无法将TDP-43重新召回至细胞核内，也不会改变GR聚集体的动态特性。综上，本研究结果支持以上调Kapβ2表达水平作为潜在新型治疗策略的设计思路…… 人类组织：来自健康对照以及携带C9ORF72内含子核苷酸重复扩增患者的人类死后中枢神经系统组织，均获取自Target ALS与Jefferson Weinberg ALS中心的生物样本库。患者与对照的信息及人口统计学资料详见表4。人类死后新鲜冰冻组织保存于-80℃环境中。 细胞培养：HEK293细胞采用DMEM培养基（Cytiva，货号：SH30243.LS）进行培养，该培养基添加10%胎牛血清（FBS，Cytiva，货号：SH30071.01HI）以及青霉素-链霉素（Thermo Fisher Scientific，货号：SV30010）。细胞每3-4天传代一次，使用0.05%胰蛋白酶（Corning，货号：25-051-C）进行消化。 原代皮层神经元：移除脑膜后，从妊娠第16天（E16）的大鼠胚胎中分离脑组织。将皮层与中脑区域剪切成小块，置于含0.2%胰蛋白酶的无钙镁HBSS培养基（Cytiva，货号：SH30588.01）中，于37℃、80rcf条件下振荡孵育45分钟，随后添加胎牛血清（Cytiva，货号：SH30071.01HI），后续实验步骤省略。 # 核输入受体Kapβ2修饰C9orf72相关ALS/FTD中poly(GR)介导的神经毒性 https://doi.org/10.5061/dryad.v41ns1s4f 本数据集包含关联论文中所有图表的源文件。 ## 数据与文件结构说明 每个标注为yx面板（y为图号，x为面板字母）的图表均配有对应的源数据。 ### 图1. GR不影响神经元中Kapβ2的表达水平 * a) 对转染GFP或GR50的皮层神经元进行Kapβ2水平的qPCR分析。数据以平均值±标准误（standard error of the mean, S.E.M.）表示（n=6次生物学重复，采用Student t检验，n.s.表示无显著性差异） * c) 该柱状图为皮层神经元提取物中Kapβ2蛋白的WB定量结果，以总蛋白染色作为内参进行归一化。数据以平均值±标准误表示（n=3次生物学重复，采用Student t检验，n.s.表示无显著性差异） * d) 对皮层与脊髓组织中Kapβ2转录水平的qPCR分析……