Chen Han-Jou, Mitchell Jacqueline C, Novoselov Sergey, Miller Jack, Nishimura Agnes L, Scotter Emma L, Vance Caroline A, Cheetham Michael E, Shaw Christopher E
Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK.
UCL Institute of Ophthalmology, 11-43 Bath Street, London, UK.
Brain. 2016 May;139(Pt 5):1417-32. doi: 10.1093/brain/aww028. Epub 2016 Mar 1.
Detergent-resistant, ubiquitinated and hyperphosphorylated Tar DNA binding protein 43 (TDP-43, encoded by TARDBP) neuronal cytoplasmic inclusions are the pathological hallmark in ∼95% of amyotrophic lateral sclerosis and ∼60% of frontotemporal lobar degeneration cases. We sought to explore the role for the heat shock response in the clearance of insoluble TDP-43 in a cellular model of disease and to validate our findings in transgenic mice and human amyotrophic lateral sclerosis tissues. The heat shock response is a stress-responsive protective mechanism regulated by the transcription factor heat shock factor 1 (HSF1), which increases the expression of chaperones that refold damaged misfolded proteins or facilitate their degradation. Here we show that manipulation of the heat shock response by expression of dominant active HSF1 results in a dramatic reduction of insoluble and hyperphosphorylated TDP-43 that enhances cell survival, whereas expression of dominant negative HSF1 leads to enhanced TDP-43 aggregation and hyperphosphorylation. To determine which chaperones were mediating TDP-43 clearance we over-expressed a range of heat shock proteins (HSPs) and identified DNAJB2a (encoded by DNAJB2, and also known as HSJ1a) as a potent anti-aggregation chaperone for TDP-43. DNAJB2a has a J domain, allowing it to interact with HSP70, and ubiquitin interacting motifs, which enable it to engage the degradation of its client proteins. Using functionally deleted DNAJB2a constructs we demonstrated that TDP-43 clearance was J domain-dependent and was not affected by ubiquitin interacting motif deletion or proteasome inhibition. This indicates that TDP-43 is maintained in a soluble state by DNAJB2a, leaving the total levels of TDP-43 unchanged. Additionally, we have demonstrated that the levels of HSF1 and heat shock proteins are significantly reduced in affected neuronal tissues from a TDP-43 transgenic mouse model of amyotrophic lateral sclerosis and patients with sporadic amyotrophic lateral sclerosis. This implies that the HSF1-mediated DNAJB2a/HSP70 heat shock response pathway is compromised in amyotrophic lateral sclerosis. Defective refolding of TDP-43 is predicted to aggravate the TDP-43 proteinopathy. The finding that the pathological accumulation of insoluble TDP-43 can be reduced by the activation of HSF1/HSP pathways presents an exciting opportunity for the development of novel therapeutics.
抗去污剂、泛素化且高度磷酸化的Tar DNA结合蛋白43（TDP - 43，由TARDBP编码）神经元胞质内含物是约95%的肌萎缩侧索硬化症和约60%的额颞叶痴呆病例中的病理标志。我们试图在疾病细胞模型中探究热休克反应在清除不溶性TDP - 43中的作用，并在转基因小鼠和人类肌萎缩侧索硬化症组织中验证我们的发现。热休克反应是一种由转录因子热休克因子1（HSF1）调节的应激反应性保护机制，它会增加伴侣蛋白的表达，这些伴侣蛋白可使受损的错误折叠蛋白重新折叠或促进其降解。在此我们表明，通过表达显性激活的HSF1来操纵热休克反应会导致不溶性和高度磷酸化的TDP - 43显著减少，从而提高细胞存活率，而表达显性负性HSF1则会导致TDP - 43聚集和磷酸化增强。为了确定哪些伴侣蛋白介导TDP - 43的清除，我们过表达了一系列热休克蛋白（HSP），并确定DNAJB2a（由DNAJB2编码，也称为HSJ1a）是TDP - 43的一种有效的抗聚集伴侣蛋白。DNAJB2a有一个J结构域，使其能够与HSP70相互作用，还有泛素相互作用基序，使其能够参与其客户蛋白的降解。使用功能缺失的DNAJB2a构建体，我们证明TDP - 43的清除依赖于J结构域，不受泛素相互作用基序缺失或蛋白酶体抑制的影响。这表明TDP - 43通过DNAJB2a维持在可溶状态，而TDP - 43的总水平不变。此外，我们已经证明，在肌萎缩侧索硬化症的TDP - 43转基因小鼠模型和散发性肌萎缩侧索硬化症患者的受影响神经元组织中，HSF1和热休克蛋白的水平显著降低。这意味着HSF1介导的DNAJB2a/HSP70热休克反应途径在肌萎缩侧索硬化症中受损。预计TDP - 43的错误折叠缺陷会加重TDP - 43蛋白病。通过激活HSF1/HSP途径可减少不溶性TDP - 43的病理积累这一发现为开发新型治疗方法提供了一个令人兴奋的机会。
