Albani Diego, Peverelli Erika, Rametta Raffaela, Batelli Sara, Veschini Lorenzo, Negro Alessandro, Forloni Gianluigi
Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
FASEB J. 2004 Nov;18(14):1713-5. doi: 10.1096/fj.04-1621fje. Epub 2004 Sep 2.
Alpha-synuclein (alpha-syn) is a 140-amino acid presinaptic protein whose mutations A30P and A53T have been linked to familiar Parkinson's disease (PD). Many data suggest that alpha-syn aggregation is the key event that triggers alpha-syn-mediated neurotoxicity. Nevertheless, other lines of evidence proposed a protective role of alpha-syn against oxidative stress (a major feature of PD), even if the exact mechanism of this protective action and the role of the pathogenetic mutations to this respect have not been elucidated yet. To address these points, we developed an in vitro model of oxidative stress by exposing PC12 cells to hydrogen peroxide (H2O2) (150 microM) for 72 h, and we evaluated alpha-syn-mediated protection delivering increasing amounts of alpha-syn (wild type [WT] or mutated) inside cells using the fusion proteins TAT-alpha-syn (WT, A30P, and A53T). We found that nanomolar amounts of TAT-alpha-syn-mediated protected against oxidative stress and other cellular injuries (6-hydroxydopamine and serum deprivation), whereas micromolar amounts of the fusion proteins were intrinsically toxic to cells. The protective effect was independent from the presence of the mutations A30P and A53T, but no protection occurred when cells were challenged with the proteasome inhibitors lactacystin and MG132. We verified that the protection mechanism required the presence of the C-terminal domain of alpha-syn, as nanomolar amounts of the C-terminal truncated fusion protein TAT-alpha-syn (WT[1-97]) failed in preventing H2O2 toxicity. To further characterize the molecular mechanisms at the basis of alpha-syn protection, we investigated the possible involvement of the chaperone protein HSP70 that is widely implicated in neuroprotection. We found that, at nanomolar concentrations, TAT-alpha-syn was able to increase HSP70 protein level, whereas at the micromolar scale, TAT-alpha-syn decreased HSP70 at the protein level. These effects on HSP70 were independent from the presence of alpha-syn pathogenetic mutations but required the alpha-syn C-terminal domain. The implications for alpha-syn-mediated neurotoxicity and for PD pathogenesis and progression are discussed.
α-突触核蛋白（α-syn）是一种由140个氨基酸组成的突触前蛋白，其A30P和A53T突变与家族性帕金森病（PD）有关。许多数据表明，α-syn聚集是引发α-syn介导的神经毒性的关键事件。然而，其他证据表明α-syn对氧化应激（PD的一个主要特征）具有保护作用，尽管这种保护作用的确切机制以及致病突变在这方面的作用尚未阐明。为了解决这些问题，我们通过将PC12细胞暴露于过氧化氢（H2O2）（150微摩尔）72小时，建立了一种氧化应激的体外模型，并使用融合蛋白TAT-α-syn（野生型[WT]或突变型）在细胞内递送增加量的α-syn（野生型或突变型），评估α-syn介导的保护作用。我们发现，纳摩尔量的TAT-α-syn介导的保护作用可抵抗氧化应激和其他细胞损伤（6-羟基多巴胺和血清剥夺），而微摩尔量的融合蛋白对细胞具有内在毒性。保护作用与A30P和A53T突变的存在无关，但当细胞用蛋白酶体抑制剂乳胞素和MG132处理时，没有发生保护作用。我们证实，保护机制需要α-syn的C末端结构域的存在，因为纳摩尔量的C末端截短融合蛋白TAT-α-syn（WT[1-97]）未能预防H2O2毒性。为了进一步表征α-syn保护作用的分子机制，我们研究了广泛参与神经保护的伴侣蛋白HSP70的可能参与情况。我们发现，在纳摩尔浓度下，TAT-α-syn能够增加HSP70蛋白水平，而在微摩尔水平上，TAT-α-syn在蛋白水平上降低HSP70。这些对HSP70的影响与α-syn致病突变的存在无关，但需要α-syn的C末端结构域。讨论了α-syn介导的神经毒性以及对PD发病机制和进展的影响。
