Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, New South Wales, Australia.
ARC Centre of Excellence in Synthetic Biology, Department of Molecular Sciences, Faculty of Science and Engineering, Macquarie University, Sydney, New South Wales, Australia.
Neuropathol Appl Neurobiol. 2021 Dec;47(7):990-1003. doi: 10.1111/nan.12749. Epub 2021 Aug 2.
Splicing factor proline and glutamine rich (SFPQ) is an RNA-DNA binding protein that is dysregulated in Alzheimer's disease and frontotemporal dementia. Dysregulation of SFPQ, specifically increased intron retention and nuclear depletion, has been linked to several genetic subtypes of amyotrophic lateral sclerosis (ALS), suggesting that SFPQ pathology may be a common feature of this heterogeneous disease. Our study aimed to investigate this hypothesis by providing the first comprehensive assessment of SFPQ pathology in large ALS case-control cohorts.
We examined SFPQ at the RNA, protein and DNA levels. SFPQ RNA expression and intron retention were examined using RNA-sequencing and quantitative PCR. SFPQ protein expression was assessed by immunoblotting and immunofluorescent staining. At the DNA level, SFPQ was examined for genetic variation novel to ALS patients.
At the RNA level, retention of SFPQ intron nine was significantly increased in ALS patients' motor cortex. In addition, SFPQ RNA expression was significantly reduced in the central nervous system, but not blood, of patients. At the protein level, neither nuclear depletion nor reduced expression of SFPQ was found to be a consistent feature of spinal motor neurons. However, SFPQ-positive ubiquitinated protein aggregates were observed in patients' spinal motor neurons. At the DNA level, our genetic screen identified two novel and two rare SFPQ sequence variants not previously reported in the literature.
Our findings confirm dysregulation of SFPQ as a pathological feature of the central nervous system of ALS patients and indicate that investigation of the functional consequences of this pathology will provide insight into ALS biology.
拼接因子脯氨酸和谷氨酸丰富（SFPQ）是一种 RNA-DNA 结合蛋白，在阿尔茨海默病和额颞叶痴呆中失调。SFPQ 的失调，特别是内含子保留和核耗竭的增加，与几种肌萎缩侧索硬化症（ALS）的遗传亚型有关，这表明 SFPQ 病理学可能是这种异质疾病的共同特征。我们的研究旨在通过对大型 ALS 病例对照队列中的 SFPQ 病理学进行全面评估来验证这一假设。
我们在 RNA、蛋白质和 DNA 水平上研究了 SFPQ。使用 RNA 测序和定量 PCR 检测 SFPQ RNA 表达和内含子保留。通过免疫印迹和免疫荧光染色评估 SFPQ 蛋白表达。在 DNA 水平上，我们检查了 ALS 患者特有的 SFPQ 遗传变异。
在 RNA 水平上，SFPQ 内含子九的保留在 ALS 患者的运动皮层中显著增加。此外，患者中枢神经系统（但血液中没有）的 SFPQ RNA 表达显著降低。在蛋白质水平上，未发现 SFPQ 核耗竭或表达减少是脊髓运动神经元的一致特征。然而，在患者的脊髓运动神经元中观察到 SFPQ 阳性泛素化蛋白聚集体。在 DNA 水平上，我们的遗传筛选确定了两个以前在文献中未报道过的 SFPQ 序列变体的两个新变体和两个罕见变体。
我们的研究结果证实 SFPQ 的失调是 ALS 患者中枢神经系统的病理特征，并表明对这种病理学的功能后果进行研究将为 ALS 生物学提供深入了解。
