Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody

Abstract

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.

eLife digest

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.