Dementia Research Group, Korea Brain Research Institute (KBRI) , Daegu, South Korea.
Soonchunhyang Institute of Medi-bio Science, Soonchunhyang University , Cheonan, South Korea.
Autophagy. 2020 Aug;16(8):1396-1412. doi: 10.1080/15548627.2019.1686729. Epub 2019 Nov 5.
TARDBP/TDP-43 (TAR DNA binding protein) proteinopathies are a common feature in a variety of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and Alzheimer disease (AD). However, the molecular mechanisms underlying TARDBP-induced neurotoxicity are largely unknown. In this study, we demonstrated that TARDBP proteinopathies induce impairment in the ubiquitin proteasome system (UPS), as evidenced by an accumulation of ubiquitinated proteins and a reduction in proteasome activity in neuronal cells. Through kinase inhibitor screening, we identified PTK2/FAK (PTK2 protein tyrosine kinase 2) as a suppressor of neurotoxicity induced by UPS impairment. Importantly, PTK2 inhibition significantly reduced ubiquitin aggregates and attenuated TARDBP-induced cytotoxicity in a model of TARDBP proteinopathies. We further identified that phosphorylation of SQSTM1/p62 (sequestosome 1) at S403 (p-SQSTM1 [S403]), a key component in the autophagic degradation of poly-ubiquitinated proteins, is increased upon TARDBP overexpression and is dependent on the activation of PTK2 in neuronal cells. Moreover, expressing a non-phosphorylated form of SQSTM1 (SQSTM1) significantly repressed the accumulation of insoluble poly-ubiquitinated proteins and neurotoxicity induced by TARDBP overexpression in neuronal cells. In addition, TBK1 (TANK binding kinase 1), a kinase that phosphorylates S403 of SQSTM1, was found to be involved in the PTK2-mediated phosphorylation of SQSTM1. Taken together, our data suggest that the PTK2-TBK1-SQSTM1 axis plays a critical role in the pathogenesis of TARDBP by regulating neurotoxicity induced by UPS impairment. Therefore, targeting the PTK2-TBK1-SQSTM1 axis may represent a novel therapeutic intervention for neurodegenerative diseases with TARDBP proteinopathies.: ALP: macroautophagy/autophagy lysosomal pathway; ALS: amyotrophic lateral sclerosis; ATXN2: ataxin 2; BafA1: bafilomycin A; cCASP3: cleaved caspase 3; CSNK2: casein kinase 2; FTLD: frontotemporal lobar degeneration; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; OPTN: optineurin; PTK2/FAK: PTK2 protein tyrosine kinase 2; SQSTM1/p62: sequestosome 1; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system.
TARDBP/TDP-43（TAR DNA 结合蛋白）蛋白病是多种神经退行性疾病的共同特征，包括肌萎缩侧索硬化症（ALS）、额颞叶变性（FTLD）和阿尔茨海默病（AD）。然而，TARDBP 诱导神经毒性的分子机制在很大程度上尚不清楚。在这项研究中，我们证明了 TARDBP 蛋白病导致泛素蛋白酶体系统（UPS）受损，这表现在神经元细胞中泛素化蛋白的积累和蛋白酶体活性的降低。通过激酶抑制剂筛选，我们鉴定出 PTK2/FAK（PTK2 蛋白酪氨酸激酶 2）是 UPS 受损诱导的神经毒性的抑制剂。重要的是，PTK2 抑制显著减少了泛素聚集体，并减轻了 TARDBP 蛋白病模型中 TARDBP 诱导的细胞毒性。我们进一步发现，在神经元细胞中，TARDBP 过表达时 SQSTM1/p62（自噬体 1）的 S403 位点（p-SQSTM1[S403]）的磷酸化增加，这是多聚泛素化蛋白自噬降解的关键成分，并且依赖于 PTK2 的激活。此外，表达非磷酸化形式的 SQSTM1（SQSTM1）可显著抑制神经元细胞中 TARDBP 过表达诱导的不溶性多聚泛素化蛋白的积累和神经毒性。此外，TBK1（TANK 结合激酶 1），一种磷酸化 SQSTM1 的 S403 位点的激酶，被发现参与了 PTK2 介导的 SQSTM1 的磷酸化。总之，我们的数据表明，PTK2-TBK1-SQSTM1 轴通过调节 UPS 受损诱导的神经毒性在 TARDBP 的发病机制中起关键作用。因此，针对 PTK2-TBK1-SQSTM1 轴可能代表了具有 TARDBP 蛋白病的神经退行性疾病的一种新的治疗干预措施。
