Decrease of neuronal FKBP4/FKBP52 modulates perinuclear lysosomal positioning and MAPT/Tau behavior during MAPT/Tau-induced proteotoxic stress

Defects of autophagy-lysosomal protein degradation are thought to contribute to the pathogenesis of several neurodegenerative diseases, and the accumulation of aggregation prone proteins such as MAPT/Tau in Alzheimer disease (AD). We previously showed the localization of the immunophilin FKBP4/FKBP52 in the lysosomal system of healthy human neurons suggesting its possible role in lysosome function. We also showed that decreased FKBP4 levels in AD brain neurons correlate with abnormal MAPT accumulation and aggregation. In this study, we demonstrate that FKBP4 decrease in a human neuronal cell line (SH-SY5Y) and in dorsal root ganglion (DRG) neurons from human MAPTP301S transgenic mice affected the function of the autophagy-lysosomal system under MAPT induced proteotoxic stress conditions. We show that acute MAPT accumulation in SH-SY5Y cells induced perinuclear clustering of lysosomes, triggered FKBP4 localization around the clusters and its colocalization with MAPT and MAP1LC3/LC3-positive autophagic vesicles; a similar FKBP4 localization was detected in some AD brain neurons. We demonstrate that FKBP4 decrease altered lysosomal clustering along with MAPT and MAP1LC3 secretion increase. Although ectopic FKBP4 expression could not induce autophagy under our experimental conditions, it prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells implying a regulatory role of FKBP4 on MAPT secretion. Finally, we observe that FKBP4 deficiency decreased MAP1LC3-II expression and provoked MAPT accumulation during long-term stress in mouse DRG neurons. We hypothesize that the abnormal FKBP4 decrease observed in AD brain neurons might hinder autophagy efficiency and contribute to the progression of the tauopathy by modulating MAPT secretion and accumulation during MAPT pathogenesis. Abbreviations: AD: Alzheimer disease; AKT/protein kinase B: AKT serine/threonine kinase; ALP: Autophagy-lysosomal pathway; ATG: autophagy-related; BafA1: bafilomycin A1; CQ: chloroquine; CTSD: cathepsin D; DIV: days in vitro; DRG: dorsal root ganglion neurons; Dox: doxycycline; DNAJC5: DnaJ heat shock protein family (Hsp40) member C5; EL: empty lentiviral vectors; ENO2/NSE: enolase 2, gamma neuronal; FKBP4/FKBP52: FKBP prolyl isomerase 4; FTLD-Tau: frontotemporal lobar degeneration with Tau pathology; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/Tau: microtubule associated protein tau; MTT: tetrazolium salt; NFTs: neurofibrillary tangles; RPE-1: retinal pigment epithelial cells; shRNA: small-hairpin ribonucleic acid; SQSTM1/p62: sequestosome 1; SD: standard deviation; SEM: standard error of the mean; SH-SY5Y: human neuroblastoma cells; Sh1 or Sh2: Lentiviral shRNA vectors inducing FKBP4 decrease; SH-52GFP: MAPT/Tau-inducible SH-SY5Y cell line constitutively expressing FKBP4-GFP; TUBB3/βIII tubulin: tubulin beta 3 class III; UPS: ubiquitin-proteasome system自噬-溶酶体蛋白降解（autophagy-lysosomal protein degradation）异常被认为参与多种神经退行性疾病的发病机制，且与阿尔茨海默病（Alzheimer disease, AD）中易聚集蛋白如微管相关蛋白tau（microtubule associated protein tau, MAPT/Tau）的蓄积密切相关。我们前期研究证实，免疫亲和素FKBP4/FKBP52定位于健康人神经元的溶酶体系统中，提示其可能在溶酶体功能调控中发挥作用。我们还发现，AD患者脑神经元中FKBP4水平下调与异常MAPT蓄积及聚集呈显著相关。 本研究中，我们证实：在人神经母细胞瘤细胞系（SH-SY5Y）以及携带人MAPTP301S突变的转基因小鼠背根神经节（dorsal root ganglion, DRG）神经元中，FKBP4表达下调会在MAPT诱导的蛋白毒性应激条件下损害自噬-溶酶体系统的功能。 我们发现，SH-SY5Y细胞中急性MAPT蓄积可诱导溶酶体发生核周聚集，触发FKBP4向聚集区域募集，并与MAPT及微管相关蛋白1轻链3（microtubule associated protein 1 light chain 3, MAP1LC3/LC3）阳性自噬囊泡发生共定位；在部分AD患者脑神经元中也检测到了类似的FKBP4定位异常。 我们证实，FKBP4下调会改变溶酶体的聚集模式，同时增加MAPT及MAP1LC3的分泌。尽管在本实验条件下，外源性FKBP4过表达无法诱导自噬发生，但可抑制SH-SY5Y细胞中MAPT蓄积后的MAPT分泌，提示FKBP4对MAPT分泌具有调控作用。 最后，我们观察到，小鼠DRG神经元在长期应激状态下，FKBP4缺失会降低MAP1LC3-II的表达水平，并诱发MAPT蓄积。我们推测，AD患者脑神经元中观察到的FKBP4异常下调可能通过调控MAPT致病过程中的MAPT分泌与蓄积，削弱自噬效率，进而促进tau蛋白病（tauopathy）的进展。 缩略语： AD：阿尔茨海默病；AKT/蛋白激酶B：AKT丝氨酸/苏氨酸激酶；ALP：自噬-溶酶体通路；ATG：自噬相关；BafA1：巴弗洛霉素A1；CQ：氯喹；CTSD：组织蛋白酶D；DIV：体外培养天数；DRG：背根神经节神经元；Dox：多西环素；DNAJC5：DnaJ热休克蛋白家族（Hsp40）成员C5；EL：空慢病毒载体；ENO2/NSE：烯醇酶2，γ神经元型（神经元特异性烯醇化酶）；FKBP4/FKBP52：FKBP脯氨酰异构酶4；FTLD-Tau：伴Tau病理的额颞叶变性；GFP：绿色荧光蛋白；LAMP1：溶酶体相关膜蛋白1；LDH：乳酸脱氢酶；MAP1LC3/LC3：微管相关蛋白1轻链3；MAPT/Tau：微管相关蛋白tau；MTT：四唑盐；NFTs：神经原纤维缠结；RPE-1：视网膜色素上皮细胞；shRNA：小发夹RNA；SQSTM1/p62：Sequestosome 1（泛素结合蛋白p62）；SD：标准差；SEM：均值标准误；SH-SY5Y：人神经母细胞瘤细胞；Sh1或Sh2：诱导FKBP4下调的慢病毒shRNA载体；SH-52GFP：组成型表达FKBP4-GFP的可诱导MAPT/Tau SH-SY5Y细胞系；TUBB3/βⅢ微管蛋白：Ⅲ类β微管蛋白；UPS：泛素-蛋白酶体系统