Department of Neuroscience, Reproductive and Odontostomatological Sciences, University of Naples Federico II, 80131 Naples, Italy.
Istituto di Scienze e Tecnologie per l'Energia e la Mobilità Sostenibili (STEMS)-CNR, 80125 Naples, Italy.
Int J Mol Sci. 2022 Oct 27;23(21):13041. doi: 10.3390/ijms232113041.
Background: An emerging body of evidence indicates an association between anthropogenic particulate matter (PM) and neurodegeneration. Although the historical focus of PM toxicity has been on the cardiopulmonary system, ultrafine PM particles can also exert detrimental effects in the brain. However, only a few studies are available on the harmful interaction between PM and CNS and on the putative pathomechanisms. Methods: Ultrafine PM particles with a diameter < 0.1 μm (PM0.1) and nanoparticles < 20 nm (NP20) were sampled in a lab-scale combustion system. Their effect on cell tracking in the space was studied by time-lapse and high-content microscopy in NSC-34 motor neurons while pHrodo™ Green conjugates were used to detect PM endocytosis. Western blotting analysis was used to quantify protein expression of lysosomal channels (i.e., TRPML1 and TPC2) and autophagy markers. Current-clamp electrophysiology and Fura2-video imaging techniques were used to measure membrane potential, intracellular Ca2+ homeostasis and TRPML1 activity in NSC-34 cells exposed to PM0.1 and NP20. Results: NP20, but not PM0.1, reduced NSC-34 motor neuron movement in the space. Furthermore, NP20 was able to shift membrane potential of motor neurons toward more depolarizing values. PM0.1 and NP20 were able to enter into the cells by endocytosis and exerted mitochondrial toxicity with the consequent stimulation of ROS production. This latter event was sufficient to determine the hyperactivation of the lysosomal channel TRPML1. Consequently, both LC3-II and p62 protein expression increased after 48 h of exposure together with AMPK activation, suggesting an engulfment of autophagy. The antioxidant molecule Trolox restored TRPML1 function and autophagy. Conclusions: Restoring TRPML1 function by an antioxidant agent may be considered a protective mechanism able to reestablish autophagy flux in motor neurons exposed to nanoparticles.
越来越多的证据表明，人为产生的颗粒物（PM）与神经退行性变之间存在关联。尽管 PM 毒性的历史重点一直是心肺系统，但超细 PM 颗粒也会对大脑造成有害影响。然而，关于 PM 与中枢神经系统的有害相互作用以及潜在的发病机制的研究还很少。方法：在实验室规模的燃烧系统中采样了直径<0.1μm（PM0.1）和<20nm（NP20）的超细 PM 颗粒。通过在 NSC-34 运动神经元中进行延时和高内涵显微镜研究，研究了它们对细胞在空间中跟踪的影响，同时使用 pHrodo™ Green 缀合物来检测 PM 的内吞作用。Western blot 分析用于定量溶酶体通道（即 TRPML1 和 TPC2）和自噬标志物的蛋白表达。采用电流钳电生理学和 Fura2 视频成像技术测量暴露于 PM0.1 和 NP20 的 NSC-34 细胞的膜电位、细胞内 Ca2+稳态和 TRPML1 活性。结果：NP20 而非 PM0.1 降低了 NSC-34 运动神经元在空间中的运动。此外，NP20 能够将运动神经元的膜电位向更去极化的方向移动。PM0.1 和 NP20 能够通过内吞作用进入细胞，并通过产生 ROS 来发挥线粒体毒性。这后一事件足以导致溶酶体通道 TRPML1 的过度激活。结果，暴露 48 小时后，LC3-II 和 p62 蛋白表达增加，同时 AMPK 激活，表明自噬的吞噬作用。抗氧化剂 Trolox 恢复了 TRPML1 功能和自噬。结论：通过抗氧化剂恢复 TRPML1 功能可能被认为是一种保护机制，能够恢复暴露于纳米颗粒的运动神经元中的自噬通量。
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