Zafar Saima, Younas Neelam, Correia Susana, Shafiq Mohsin, Tahir Waqas, Schmitz Matthias, Ferrer Isidre, Andréoletti Olivier, Zerr Inga
Department of Neurology, Clinical Dementia Center and DZNE, University Medical Centre Goettingen, 37075, Goettingen, Germany.
Institute of Neuropathology, IDIBELL-University Hospital Bellvitge, University of Barcelona, Hospitalet de Llobregat, Spain.
Mol Neurobiol. 2017 Jan;54(1):697-709. doi: 10.1007/s12035-016-9694-8. Epub 2016 Jan 14.
There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease AD brain. In this study, we performed a targeted search for endocytic pathway proteins in the different regions of the brain. We found late endosome marker Rab7a which was significantly upregulated in the frontal cortex region in the rapid progressive CJD form (MM1) and rapid progressive AD (rpAD) forms. However, Rab9 expression was significantly downregulated only in CJD-MM1 brain frontal cortex region. In the cerebellum, Rab7a expression showed significant upregulation in both subtype MM1 and VV2 CJD forms, in contrast to Rab9 which showed significant downregulation in both subtype MM1 and VV2 CJD forms at terminal stage of the disease. To check regulatory response at pre-symptomatic stage of the disease, we checked the regulatory interactive response of Rab7a, Rab9, and known biomarkers PrP and tau forms in frontal cortex at pre-symptomatic stage of the disease in tg340 mice expressing about fourfold of human PrP-M129 with PrP-null background that had been inoculated with human sCJD MM1 brain tissue homogenates (sCJD MM1 mice). In addition, we analyzed 5XFAD mice, exhibiting five mutations in the APP and presenilin genes related to familial Alzheimer's disease (FAD), to validate specific regulatory response of Rab7a, Rab9, tau, and phosphorylated form of tau by immunostaining 5XFAD mice in comparison with the wild-type age-matched mice brain. The cortical region of 5XFAD mice brain showed accumulated form of Rab7a in puncta that co-label for p-Tau, indicating colocalization by using confocal laser-scanning microscopy and was confirmed by using reverse co-immunoprecipitation. Furthermore, synthetic RNA (siRNA) against the Rab7a gene decreased expression of Rab7a protein, in cortical primary neuronal cultures of PrP wild type. This depleted expression of Rab7a led to the increased accumulation of PrP in Rab9-positive endosomal compartments and consequently an increased co-localization between PrP/Rab9; however, total tau level decreased. Interestingly, siRNA against tau gene in cortical primary neuronal cultures of PrP wild-type mice showed enhanced Rab7a and Rab9 expression and increase formation of dendritic spines. The work described highlighted the selective involvement of late endosomal compartment marker Rab7a in CJD, slow and rapid progressive forms of AD pathogenesis.
对神经退行性疾病早期病理生理学的理解需求日益增加。内吞机制和细胞骨架相关反应的变化是在克雅氏病（CJD）和阿尔茨海默病（AD）大脑中观察到的首批改变。在本研究中，我们针对性地搜索了大脑不同区域的内吞途径蛋白。我们发现晚期内体标志物Rab7a在快速进展型CJD（MM1型）和快速进展型AD（rpAD型）的额叶皮质区域显著上调。然而，Rab9的表达仅在CJD-MM1型大脑额叶皮质区域显著下调。在小脑中，Rab7a的表达在MM1型和VV2型CJD中均显著上调，相反，Rab9在疾病终末期的MM1型和VV2型CJD中均显著下调。为了检查疾病症状前阶段的调节反应，我们在表达约四倍人类PrP-M129且PrP基因缺失背景的tg340小鼠中，检查了疾病症状前阶段额叶皮质中Rab7a、Rab9以及已知生物标志物PrP和tau形式的调节相互作用反应，这些小鼠已接种人类sCJD MM1脑组织匀浆（sCJD MM1小鼠）。此外，我们分析了5XFAD小鼠，其APP和早老素基因存在与家族性阿尔茨海默病（FAD）相关的五个突变，通过免疫染色5XFAD小鼠并与年龄匹配的野生型小鼠大脑进行比较，以验证Rab7a、Rab9、tau以及tau的磷酸化形式的特异性调节反应。5XFAD小鼠大脑的皮质区域显示Rab7a以点状形式积累，与p-Tau共标记，通过共聚焦激光扫描显微镜表明其共定位，并通过反向共免疫沉淀得到证实。此外，针对Rab7a基因的合成RNA（siRNA）降低了PrP野生型皮质原代神经元培养物中Rab7a蛋白的表达。Rab7a表达的这种减少导致PrP在Rab9阳性内体区室中的积累增加，从而导致PrP/Rab9之间的共定位增加；然而，总tau水平降低。有趣的是，针对PrP野生型小鼠皮质原代神经元培养物中tau基因的siRNA显示Rab7a和Rab9表达增强以及树突棘形成增加。所描述的工作突出了晚期内体区室标志物Rab7a在CJD、AD发病机制的缓慢和快速进展形式中的选择性参与。
