Adamson D C, Kopnisky K L, Dawson T M, Dawson V L
Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
J Neurosci. 1999 Jan 1;19(1):64-71. doi: 10.1523/JNEUROSCI.19-01-00064.1999.
Of the individuals with human immunodeficiency virus type 1 (HIV-1) infection, 20-30% will develop the neurological complication of HIV-associated dementia (HAD). The mechanisms underlying HAD are unknown; however, indirect immunologically mediated mechanisms are theorized to play a role. Recently, the HIV-1 coat protein gp41 has been implicated as a major mediator of HAD through induction of neurocytokines and subsequent neuronal cell death. Using primary mixed cortical cultures from neuronal nitric oxide synthase (NOS) null (nNOS-/-) mice and immunological NOS null (iNOS-/-) mice, we establish iNOS-derived NO as a major mediator of gp41 neurotoxicity. Neurotoxicity elicited by gp41 is markedly attenuated in iNOS-/- cultures compared with wild-type and nNOS-/- cultures. The NOS inhibitor L-nitroarginine methyl ester is neuroprotective in wild-type and nNOS-/- cultures, confirming the role of iNOS-derived NO in gp41 neurotoxicity. Confirming that iNOS-/- cultures lack iNOS, gp41 did not induce iNOS in iNOS-/- cultures, but it markedly induced iNOS in wild-type and nNOS-/- cultures. We elucidate the region of gp41 that is critical for iNOS induction and neuronal cell death by monitoring iNOS induction with overlapping peptides spanning gp41. We show that the N-terminal region of gp41, which we designate as the neurotoxic domain, induces iNOS protein activity and iNOS-dependent neurotoxicity at picomolar concentrations in a manner similar to recombinant gp41 protein. Our experiments suggest that gp41 is eliciting the induction of iNOS through potential cell surface receptors or binding sites because the induction of iNOS is dose dependent and saturable and occurs at physiologically relevant concentrations. These data confirm that the induction of iNOS by gp41 and the production of NO are primary mediators of neuronal damage and identify a neurotoxic domain of gp41 that may play an important role in HAD.
在感染1型人类免疫缺陷病毒（HIV-1）的个体中，20%至30%会出现与HIV相关痴呆（HAD）的神经并发症。HAD的潜在机制尚不清楚；然而，据推测间接免疫介导机制发挥了作用。最近，HIV-1包膜蛋白gp41被认为是HAD的主要介质，它通过诱导神经细胞因子并随后导致神经元细胞死亡而起作用。利用来自神经元型一氧化氮合酶（NOS）基因敲除（nNOS-/-）小鼠和免疫型NOS基因敲除（iNOS-/-）小鼠的原代混合皮质培养物，我们确定诱导型一氧化氮合酶（iNOS）衍生的一氧化氮（NO）是gp41神经毒性的主要介质。与野生型和nNOS-/-培养物相比，gp41在iNOS-/-培养物中引起的神经毒性明显减弱。NOS抑制剂L-硝基精氨酸甲酯在野生型和nNOS-/-培养物中具有神经保护作用，证实了iNOS衍生的NO在gp41神经毒性中的作用。通过确认iNOS-/-培养物中缺乏iNOS，gp41在iNOS-/-培养物中未诱导iNOS，但在野生型和nNOS-/-培养物中显著诱导了iNOS。我们通过用跨越gp41的重叠肽监测iNOS的诱导来阐明gp41中对iNOS诱导和神经元细胞死亡至关重要的区域。我们表明，gp41的N端区域（我们将其指定为神经毒性结构域）以类似于重组gp41蛋白的方式在皮摩尔浓度下诱导iNOS蛋白活性和iNOS依赖性神经毒性。我们的实验表明，gp41通过潜在的细胞表面受体或结合位点诱导iNOS，因为iNOS的诱导是剂量依赖性的且可饱和的，并且在生理相关浓度下发生。这些数据证实了gp41诱导iNOS和产生NO是神经元损伤的主要介质，并确定了gp41的一个神经毒性结构域，其可能在HAD中起重要作用。
