背景:小胶质细胞是大脑中的主要吞噬细胞,可以诱导神经炎症。此外,它们对α-突触核蛋白(α-syn)聚集和增殖至关重要。据报道,来自诊断为帕金森氏病(PD-exo)的患者的血浆外泌体诱发了小胶质细胞的α-syn聚集和炎症。反过来,小胶质细胞内化并释放外泌体α-syn,增强α-syn的传播。然而,PD-exo影响α-syn降解的具体机制尚不清楚。
方法:采用差分法从PD患者血浆中提取外泌体,使用电子显微镜(EM)和纳米颗粒流式细胞术进行分析,并立体定向注射到小鼠的单侧纹状体中。透射EM用于可视化BV2细胞中的溶酶体和自噬体,和溶酶体pH用LysoSensor黄/蓝DND-160测量。组织蛋白酶B和D,溶酶体相关膜蛋白1(LAMP1),ATP6V1G1,肿瘤易感基因101蛋白,calnexin,α-syn,电离钙结合衔接分子1和NLR家族pyrin结构域包含3使用定量聚合酶链反应或蛋白质印迹进行评估,和α-syn,还通过免疫荧光观察到LAMP1和ATP6V1G1。使用Lipofectamine®3000将针对V1G1的小干扰核糖核酸转染到BV2细胞和原代小胶质细胞中。通过向小鼠注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)建立PD小鼠模型。采用慢病毒介导的策略在MPTP处理的小鼠的脑中过表达ATP6V1G1。使用旋转杆和极点测试评估电机协调,使用免疫荧光组织化学和酪氨酸羟化酶的蛋白质印迹法确定小鼠黑质和纹状体组织中的神经变性。
结果:PD-exo降低了V1G1的表达,负责细胞内和细胞外环境的酸化。溶酶体酸化的这种损害导致溶酶体异常肿胀和溶酶体酶活性降低的积累。损害溶酶体蛋白降解并引起α-syn积累。此外,V1G1过表达在MPTP暴露期间赋予小鼠神经保护作用。
结论:致病蛋白积累是PD的关键特征,受损的V型ATP酶功能障碍可能参与PD的发病机制。此外,在基于MPTP的PD小鼠模型中,V1G1过表达可防止神经元毒性,这可能为开发PD治疗的新型治疗干预措施提供机会。
Microglia are the main phagocytes in the brain and can induce neuroinflammation. Moreover, they are critical to alpha-synuclein (α-syn) aggregation and propagation. Plasma exosomes derived from patients diagnosed with Parkinson\'s disease (PD-exo) reportedly evoked α-syn aggregation and inflammation in microglia. In turn, microglia internalized and released exosomal α-syn, enhancing α-syn propagation. However, the specific mechanism through which PD-exo influences α-syn degradation remains unknown.
Exosomes were extracted from the plasma of patients with PD by differential ultracentrifugation, analyzed using electron microscopy (EM) and nanoparticle flow cytometry, and stereotaxically injected into the unilateral striatum of the mice. Transmission EM was employed to visualize lysosomes and autophagosomes in BV2 cells, and lysosome pH was measured with LysoSensor Yellow/Blue DND-160. Cathepsin B and D, lysosomal-associated membrane protein 1 (LAMP1), ATP6V1G1, tumor susceptibility gene 101 protein, calnexin, α-syn, ionized calcium binding adaptor molecule 1, and NLR family pyrin domain containing 3 were evaluated using quantitative polymerase chain reaction or western blotting, and α-syn, LAMP1, and ATP6V1G1 were also observed by immunofluorescence. Small interfering ribonucleic acid against V1G1 was transfected into BV2 cells and primary microglia using Lipofectamine® 3000. A PD mouse model was established via injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into mice. A lentiviral-mediated strategy to overexpress ATP6V1G1 in the brain of MPTP-treated mice was employed. Motor coordination was assessed using rotarod and pole tests, and neurodegeneration in the mouse substantia nigra and striatum tissues was determined using immunofluorescence histochemical and western blotting of tyrosine hydroxylase.
PD-exo decreased the expression of V1G1, responsible for the acidification of intra- and extracellular milieu. This impairment of lysosomal acidification resulted in the accumulation of abnormally swollen lysosomes and decreased lysosomal enzyme activities, impairing lysosomal protein degradation and causing α-syn accumulation. Additionally, V1G1 overexpression conferred the mice neuroprotection during MPTP exposure.
Pathogenic protein accumulation is a key feature of PD, and compromised V-type ATPase dysfunction might participate in PD pathogenesis. Moreover, V1G1 overexpression protects against neuronal toxicity in an MPTP-based PD mouse model, which may provide opportunities to develop novel therapeutic interventions for PD treatment.