Paxinou E, Chen Q, Weisse M, Giasson B I, Norris E H, Rueter S M, Trojanowski J Q, Lee V M, Ischiropoulos H
Stokes Research Institute and Department of Biochemistry and Biophysics, Children's Hospital of Philadelphia and The University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Neurosci. 2001 Oct 15;21(20):8053-61. doi: 10.1523/JNEUROSCI.21-20-08053.2001.
Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.
含有丝状和聚集性α-突触核蛋白的脑损伤是神经退行性突触核蛋白病的标志。氧化应激与这些损伤的形成有关。利用稳定转染野生型和突变型α-突触核蛋白的HEK 293细胞，我们证明细胞内硝化剂的产生会导致α-突触核蛋白聚集体的形成。细胞同时暴露于产生一氧化氮和超氧化物的化合物中，通过监测二氢罗丹明123的氧化和α-突触核蛋白的硝化来证明细胞内过氧亚硝酸盐的形成。使用抗α-突触核蛋白抗体的光学显微镜和电子显微镜显示，在产生过氧亚硝酸盐的条件下，细胞核周有聚集体存在，但当细胞分别暴露于产生一氧化氮或超氧化物的化合物时则没有。在表达野生型或A53T突变型α-突触核蛋白的细胞中有20%-30%观察到α-突触核蛋白聚集体，在表达A30P突变型α-突触核蛋白的细胞中有5%观察到。在未转染的细胞或表达β-突触核蛋白的细胞中未观察到突触核蛋白聚集的证据。相反，蛋白酶体的选择性抑制导致在大多数未转染的细胞和表达α-突触核蛋白的细胞中形成用泛素抗体检测到的聚集体。然而，α-突触核蛋白与这些聚集体不共定位，表明蛋白酶体的抑制并不促进α-突触核蛋白聚集。此外，蛋白酶体抑制并未改变α-突触核蛋白的稳态水平，但添加溶酶体促渗剂氯化铵显著增加了α-突触核蛋白的量，表明溶酶体参与α-突触核蛋白的降解。我们的数据表明，硝化和氧化损伤可能引发α-突触核蛋白聚集体的发病机制。
