Cryptic exon splicing function of TARDBP interacts with autophagy in nervous tissue

TARDBP (TAR DNA binding protein) is one of the components of neuronal aggregates in sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. We have developed a simple quantitative method to evaluate TARDBP splicing function that was applied to spinal cord, brainstem, motor cortex, and occipital cortex in ALS (n = 8) cases compared to age- and gender-matched control (n = 17). Then, we quantified the abundance of a TARDBP-spliced cryptic exon present in <i>ATG4B</i> (autophagy related 4B cysteine peptidase) mRNA. Results of these analyses demonstrated that the loss of this <i>TARDBP</i> function in spinal cord, brainstem, motor cortex, and occipital cortex differentiated ALS from controls (area under the curve of receiver operating characteristic: 0.85). Significant correlations were also observed between cryptic exon levels, age, disease duration, and aberrant mRNA levels. To test if <i>TARDBP</i> function in splicing is relevant in <i>ATG4B</i> major function (autophagy) we downregulated <i>TARDBP</i> expression in human neural tissue and in HeLa cells, demonstrating that TARDBP is required for maintaining the expression of <i>ATG4B</i>. Further, <i>ATG4B</i> overexpression alone is sufficient to completely prevent the increase of SQSTM1 induced by <i>TARDBP</i> downregulation in human neural tissue cells and in cell lines. In conclusion, the present findings demonstrate abnormal alternative splicing of <i>ATG4B</i> transcripts in ALS neural tissue in agreement with <i>TARDBP</i> loss of function, leading to impaired autophagy. <b>Abbreviations</b>: ALS: amyotrophic lateral sclerosis; <i>ATG4B</i>: autophagy related 4B cysteine peptidase; AUC: area under the curve; FTLD: frontotemporal lobar degeneration; iPSC: induced pluripotent stem cells; ROC: receiver operating characteristic; <i>TARDBP</i>: TAR DNA binding protein; RT-qPCR: quantitative RT-PCR

TAR DNA结合蛋白（TARDBP）是散发性肌萎缩侧索硬化（ALS）与额颞叶变性（FTLD）患者神经元聚集体的组成成分之一。本研究开发了一种简便的定量方法以评估TARDBP的剪接功能，并将其应用于8例ALS患者（n = 8）的脊髓、脑干、运动皮层及枕叶皮层，同时以17例年龄、性别匹配的对照个体（n = 17）作为参照。随后，我们对<i>自噬相关4B半胱氨酸肽酶（ATG4B）</i> mRNA中存在的TARDBP剪接隐性外显子的丰度进行了定量检测。上述分析结果显示，ALS患者脊髓、脑干、运动皮层及枕叶皮层中TARDBP功能缺失可有效区分患者与对照个体，其受试者工作特征曲线下面积（AUC）可达0.85。此外，隐性外显子水平与年龄、病程及异常mRNA水平之间均存在显著相关性。为验证TARDBP的剪接功能是否与<i>ATG4B</i>的核心功能（自噬）相关，我们分别在人神经组织与海拉（HeLa）细胞中下调了TARDBP的表达，结果证实TARDBP是维持<i>ATG4B</i>表达的必需因子。进一步研究表明，仅过表达<i>ATG4B</i>即可完全阻断人神经组织细胞及细胞系中因TARDBP下调所诱导的SQSTM1水平升高。综上，本研究结果证实，ALS患者神经组织中<i>ATG4B</i>转录本的异常可变剪接与TARDBP功能缺失相符，最终导致自噬功能受损。**缩写**：ALS：肌萎缩侧索硬化；<i>ATG4B</i>：自噬相关4B半胱氨酸肽酶；AUC：受试者工作特征曲线下面积；FTLD：额颞叶变性；iPSC：诱导多能干细胞；ROC：受试者工作特征；TARDBP：TAR DNA结合蛋白；RT-qPCR：定量逆转录聚合酶链反应