Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody.
Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody.
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DOI：
10.7554/elife.36217
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发表时间：
2018-08-03
期刊：
影响因子：
7.7
通讯作者：
Nicke A
中科院分区：
文献类型：
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作者：
Kaczmarek-Hajek K;Zhang J;Kopp R;Grosche A;Rissiek B;Saul A;Bruzzone S;Engel T;Jooss T;Krautloher A;Schuster S;Magnus T;Stadelmann C;Sirko S;Koch-Nolte F;Eulenburg V;Nicke A
P2 X7通道参与多种CNS疾病的发病机制。越来越多的研究表明它存在于神经元中，其假定的功能仍然存在争议超过十年。为了解决这个问题，并提供一个模型，用于分析P2 X7的功能，我们产生了P2 X7 BAC转基因小鼠，允许可视化的功能性EGFP标记的P2 X7受体在体内。对这些小鼠的广泛表征揭示了小胶质细胞、Bergmann胶质细胞和少突胶质细胞中的显性P2 X7-EGFP蛋白表达，但在神经元中不表达。这些发现通过小胶质细胞和少突胶质细胞特异性P2 X7缺失和新型P2 X7特异性纳米抗体得到进一步验证。除了第一次定量分析P2 X7蛋白在中枢神经系统中的表达，我们显示其过度表达在缺血性视网膜和创伤后大脑皮质灰质的潜在后果。这种新的小鼠模型克服了P2 X7研究的局限性，将有助于确定其生理作用和对疾病的贡献。人体依赖一种叫做ATP的分子作为能量来源和信使。当细胞死亡时，例如，如果它们被损坏或由于炎症，它们会释放大量的ATP到它们的环境中。它们的邻居可以通过特定的受体检测ATP的分泌，这些受体是位于细胞表面并可以结合外部试剂的蛋白质。科学家们认为，这些ATP结合受体之一P2 X7通过触发导致炎症和细胞死亡的级联反应来响应高水平的ATP。P2 X7似乎也在癫痫和阿尔茨海默氏症等几种脑部疾病中发挥作用，但确切的机制尚不清楚。特别是，这种受体如何参与神经元的死亡尚不清楚，研究人员仍在争论P2 X7是否存在于神经元和其他类型的脑细胞中。为了回答这个问题，Kaczmarek-Hájek，Zhang，Kopp等人创造了转基因小鼠，其中P2 X7受体携带荧光染料。强大的显微镜可以从染料中拾取光信号，并帮助揭示哪些细胞具有受体。这些实验表明，神经元不携带这种蛋白质;相反，P2 X7存在于某些保持神经元健康的脑细胞中。例如，它存在于“清理”器官的免疫细胞以及支持和隔离神经元的细胞中。Kaczmarek-Hájek等人进一步提供的初步数据表明，在某些条件下，如果这些细胞中存在太多的P2 X7受体，神经元损伤可能会增加。因此，当大量ATP释放时，携带P2 X7的脑细胞可能间接导致神经元死亡。设计用于实验的基因工程小鼠可用于进一步研究，以剖析P2 X7在神经系统疾病中的作用。特别是，这种小鼠模型可能有助于了解受体是否可以成为神经退行性疾病的药物靶点。
The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7 BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors in vivo. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases. The human body relies on a molecule called ATP as an energy source and as a messenger. When cells die, for example if they are damaged or because of inflammation, they release large amounts of ATP into their environment. Their neighbors can detect the outpouring of ATP through specific receptors, the proteins that sit at the cell’s surface and can bind external agents. Scientists believe that one of these ATP-binding receptors, P2X7, responds to high levels of ATP by triggering a cascade of reactions that results in inflammation and cell death. P2X7 also seems to play a role in several brain diseases such as epilepsia and Alzheimer’s, but the exact mechanisms are not known. In particular, how this receptor is involved in the death of neurons is unclear, and researchers still debate whether P2X7 is present in neurons and in other types of brain cells. To answer this, Kaczmarek-Hájek, Zhang, Kopp et al. created genetically modified mice in which the P2X7 receptors carry a fluorescent dye. Powerful microscopes can pick up the light signal from the dye and help to reveal which cells have the receptors. These experiments show that neurons do not carry the protein; instead, P2X7 is present in certain brain cells that keep the neurons healthy. For example, it is found in the immune cells that ‘clean up’ the organ, and the cells that support and insulate neurons. Kaczmarek-Hájek et al. further provide preliminary data suggesting that, under certain conditions, if too many P2X7 receptors are present in these cells neuronal damage might be increased. It is therefore possible that the brain cells that carry P2X7 indirectly contribute to the death of neurons when large amounts of ATP are released. The genetically engineered mouse designed for the experiments could be used in further studies to dissect the role that P2X7 plays in diseases of the nervous system. In particular, this mouse model might help to understand whether the receptor could become a drug target for neurodegenerative conditions.