Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle Upon Tyne, NE2 4HH, UK.
Newcastle University Protein and Proteome Analysis Unit, Newcastle University, Newcastle Upon Tyne, UK.
Acta Neuropathol Commun. 2022 Jul 6;10(1):98. doi: 10.1186/s40478-022-01403-x.
Dementia with Lewy bodies (DLB) is pathologically defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neurons in the brain. Predominately pre-synaptic, aSyn has been reported in various subcellular compartments in experimental models. Indeed, nuclear alpha-synuclein (aSyn) is evident in many models, the dysregulation of which is associated with altered DNA integrity, transcription and nuclear homeostasis. However, the presence of aSyn in human brain cells remains controversial, yet the determination of human brain aSyn and its pathological modification is essential for understanding synucleinopathies. Here, using a multi-disciplinary approach employing immunohistochemistry, immunoblot, and mass-spectrometry (MS), we confirm aSyn in post-mortem brain tissue obtained from DLB and control cases. Highly dependent on antigen retrieval methods, in optimal conditions, intra-nuclear pan and phospho-S129 positive aSyn puncta were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations in all cases examined. Furthermore, an increase in nuclear phospho-S129 positive aSyn immunoreactivity was apparent in DLB cases compared to controls, in both neuronal and non-neuronal cell types. Our initial histological investigations identified that aSyn is affected by epitope unmasking methods but present under optimal conditions, and this presence was confirmed by isolation of nuclei and a combined approach of immunoblotting and mass spectrometry, where aSyn was approximately tenfold less abundant in the nucleus than cytoplasm. Notably, direct comparison of DLB cases to aged controls identified increased pS129 and higher molecular weight species in the nuclei of DLB cases, suggesting putative pathogenic modifications to aSyn in DLB. In summary, using multiple approaches we provide several lines of evidence supporting the presence of aSyn in autoptic human brain tissue and, notably, that it is subject to putative pathogenic modifications in DLB that may contribute to the disease phenotype.
路易体痴呆症（DLB）在病理学上定义为脑内神经元中α-突触核蛋白（aSyn）的细胞质积累。aSyn 主要存在于突触前，在实验模型中已在各种亚细胞隔室中报道。事实上，核内 α-突触核蛋白（aSyn）在许多模型中都很明显，其失调与改变 DNA 完整性、转录和核内稳态有关。然而，aSyn 在人类脑细胞中的存在仍然存在争议，但确定人类大脑中的 aSyn 及其病理修饰对于理解突触核蛋白病至关重要。在这里，我们使用多学科方法，包括免疫组织化学、免疫印迹和质谱（MS），确认了从 DLB 和对照病例中获得的死后脑组织中的 aSyn。高度依赖于抗原回收方法，在最佳条件下，在固定脑切片中的皮质神经元和非神经元细胞以及所有检查的核分离物中观察到核内泛和磷酸化 S129 阳性 aSyn 点状。此外，与对照组相比，DLB 病例中的核磷酸化 S129 阳性 aSyn 免疫反应性明显增加，无论是在神经元和非神经元细胞类型中。我们的初步组织学研究表明，aSyn 受表位暴露方法的影响，但在最佳条件下存在，这一点通过核分离和免疫印迹和质谱的联合方法得到了证实，其中 aSyn 在核内的丰度比细胞质中约低十倍。值得注意的是，将 DLB 病例与老年对照组直接比较，发现 DLB 病例核中 pS129 和更高分子量的物质增加，表明 DLB 中 aSyn 存在潜在的致病修饰。总之，我们使用多种方法提供了几条证据，支持 aSyn 存在于尸检人脑组织中，并且值得注意的是，它可能会受到 DLB 中潜在的致病修饰的影响，从而导致疾病表型。
