Abstract

Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinson’s disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA-related PD/DLB (GBA-PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA-PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.

中文翻译：

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偶发性、散发性和 GBA 相关路易体病受试者黑质神经元中 TFEB 亚细胞定位的改变

摘要

转录因子 EB (TFEB) 是参与维持自噬和溶酶体稳态的基因的主要调节因子，这些过程与GBA相关和散发性帕金森病 (PD) 和路易体痴呆 (DLB) 的发病机制有关。 TFEB 激活导致其从细胞质易位至细胞核。在这里，我们研究了 TFEB 亚细胞定位及其与偶发路易体病 (iLBD)、 GBA相关 PD/DLB ( GBA -PD/DLB)个体死后人脑中细胞内 α-突触核蛋白 (aSyn) 积累的关系或散发性 PD/DLB (sPD/DLB)，与对照受试者相比。我们使用高分辨率共聚焦和受激发射损耗（STED）显微镜分析黑质多巴胺能神经元，并对 TFEB 亚细胞定位模式进行半定量评分。我们观察到，在散发性和GBA相关病例以及 iLBD 病例中，与对照组相比，PD/DLB 患者的核 TFEB 免疫反应性降低。在所有组中，具有 Ser129 磷酸化 (pSer129) aSyn 积累的神经元中 TFEB 的核耗竭更为明显。重要的是，我们在位于高尔基体的人类多巴胺能神经元中观察到了先前未识别的 TFEB 免疫阳性核周簇。与对照组相比，这些 TFEB 簇在 iLBD、sPD/DLB 和GBA -PD/DLB中观察到更频繁且更严重，特别是在 pSer129 aSyn 阳性神经元中，但也在缺乏可检测到的 aSyn 积累的神经元中。在aSyn阴性细胞中，GBA -PD/DLB和iLBD患者中更频繁地观察到细胞质TFEB簇，并且与GBA酶活性降低以及Braak LB阶段增加相关。 TFEB 分布的改变伴随着选定的 TFEB 调节基因的总体 mRNA 表达水平的降低，表明溶酶体调节可能存在早期功能障碍。总体而言，我们在 iLBD 和 PD/DLB 患者中观察到细胞质 TFEB 在高尔基体中的保留和积累，而没有明显的 pSer129 aSyn 积累。这表明细胞疾病早期阶段可能存在 TFEB 损伤，并强调 TFEB 作为突触核蛋白病的有希望的治疗靶点。