Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Canada.
Glia. 2010 Mar;58(4):446-57. doi: 10.1002/glia.20936.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP-1-induced depletion of cellular NAD(+), glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP-dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP-1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP-1 activation by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC(50) = 78.2 +/- 2.7 microM). HPLC revealed MNNG-induced time-dependent reductions in NAD(+) (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [(3)H]glutamate uptake rate (V(max)) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 +/- 2.8 in control cells to 2.1 +/- 0.8 nmol/min/mg protein 4 h post-MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP-1, directly indicating PARP-1 involvement, and by adding exogenous NAD(+) (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co-culture with PARP-1(-/-) astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP-1 can raise glutamate levels sufficiently to have receptor-mediated effects at neighboring neurons. Taken together, these experiments showed that PARP-1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity.
聚（ADP-核糖）聚合酶 1（PARP-1）是一种普遍存在的核酶，参与基因组稳定性。过多的氧化 DNA 链断裂导致 PARP-1 诱导的细胞 NAD（+）耗竭、糖酵解速率、ATP 水平和最终细胞死亡。谷氨酸神经递质的传递受到 ATP 依赖性星形胶质细胞谷氨酸转运体的严格控制，因此我们假设 DNA 损伤导致星形胶质细胞 PARP-1 激活导致生物能量耗竭和谷氨酸摄取受损。DNA 烷化剂 N-甲基-N'-硝基-N-亚硝基胍（MNNG）激活 PARP-1 导致培养的皮质星形胶质细胞存活率显著降低（EC（50）=78.2 +/- 2.7 microM）。HPLC 显示 MNNG 诱导的 NAD（+）（98%，4 h）、ATP（71%，4 h）、ADP（63%，4 h）和 AMP（66%，4 h）时间依赖性减少。最大 [(3)H]谷氨酸摄取率（V（max））也以与 ATP 耗竭时间相关的方式下降，从对照细胞中的 19.3 +/- 2.8 下降到 MNNG 后 4 小时的 2.1 +/- 0.8 nmol/min/mg 蛋白。通过 PARP-1 的基因缺失直接表明 PARP-1 的参与，以及通过添加外源性 NAD（+）（10 mM），都可以减轻生物能量耗竭和谷氨酸摄取能力的丧失。在混合神经元/星形胶质细胞培养物中，MNNG 神经毒性部分由细胞外谷氨酸介导，并通过与 PARP-1（-/-）星形胶质细胞共培养而减轻，表明 PARP-1 对星形胶质细胞谷氨酸摄取的损害足以使相邻神经元产生受体介导的效应。总之，这些实验表明，PARP-1 激活导致星形胶质细胞总腺嘌呤核苷酸池耗竭，并严重降低神经保护谷氨酸摄取能力。
