Daigle J Gavin, Krishnamurthy Karthik, Ramesh Nandini, Casci Ian, Monaghan John, McAvoy Kevin, Godfrey Earl W, Daniel Dianne C, Johnson Edward M, Monahan Zachary, Shewmaker Frank, Pasinelli Piera, Pandey Udai Bhan
Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA, USA.
Division of Child Neurology, Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
Acta Neuropathol. 2016 Apr;131(4):605-20. doi: 10.1007/s00401-015-1530-0. Epub 2016 Jan 4.
Amyotrophic lateral sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA-binding proteins, have been linked to ALS pathology. Recently, Pur-alpha, a DNA/RNA-binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA-mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics.
肌萎缩侧索硬化症的特征是大脑和脊髓中的运动神经元进行性丧失。包括FUS、TDP43、Matrin 3、hnRNPA2和其他RNA结合蛋白在内的多个基因的突变与肌萎缩侧索硬化症的病理相关。最近，发现一种DNA/RNA结合蛋白Pur-alpha可与C9orf72重复扩增序列结合，并可能在肌萎缩侧索硬化症的发病机制中起作用。在果蝇和哺乳动物细胞培养模型中，Pur-alpha过表达时可减轻与脆性X肿瘤共济失调综合征（FXTAS）和C9orf72重复扩增疾病相关的毒性。然而，Pur-alpha在调节肌萎缩侧索硬化症发病机制中的功能尚未完全了解。我们在携带FUS致病突变的肌萎缩侧索硬化症患者细胞中，将Pur-alpha鉴定为细胞质应激颗粒（SGs）的一种新成分。当细胞受到应激挑战时，我们观察到Pur-alpha在肌萎缩侧索硬化症患者细胞中与突变型FUS共定位，并被困在组成性应激颗粒中。我们还发现FUS在哺乳动物神经元细胞中与Pur-alpha发生物理相互作用。有趣的是，shRNA介导的内源性Pur-alpha敲低显著减少了哺乳动物细胞中细胞质应激颗粒的形成，这表明Pur-alpha对SGs的形成至关重要。此外，Pur-alpha的异位表达阻止了突变型FUS的细胞质错误定位，并强烈抑制了原代运动神经元中与突变型FUS表达相关的毒性。我们的数据强调了应激颗粒在肌萎缩侧索硬化症发病机制中的重要性，并将Pur-alpha鉴定为SGs动态变化的一种新型调节因子。
