Abstract
A large body of evidence suggests the involvement of the ATP-gated purinergic receptor P2X7 (P2X7R) in neurodegenerative diseases, including Alzheimer’s disease. While it is well-described to be present and functional on microglia cells contributing to inflammatory responses, some reports suggest a neuronal expression of the receptor as well. Here, we present experimental results showing P2X7 receptors to be expressed on human hiPSC-derived microglia-like cells, hiPSC-derived neuronal progenitors and hiPSC-derived matured neuronal cells. By applying cell surface protein detection assays, we show that P2X7R is not localized on the cell membrane, despite being detected in neuronal cells and thus may not be available for directly mediating neurotoxicity. On hiPSC-derived microglia-like cells, a clear membranous expression was detected. Additionally, we have not observed differences in P2X7R functions between control and familial Alzheimer’s disease patient-derived neuronal cells. Functional assays employing a P2X7R antagonist JNJ 47965567 confirm these findings by showing P2X7R-dependent modulation of microglia-like cells viability upon treatment with P2X7R agonists ATP and BzATP, while the same effect was absent from neuronal cells. Since the majority of P2X7R research was done on rodent models, our work on human hiPSC-derived cells presents a valuable contribution to the field, extending the work on animal models to the human cellular system and toward clinical translation.
Highlights
Neuroinflammation is one of the major underlying pathologies of many neuropathological conditions, including Alzheimer’s disease (AD)
The neuronal cells were kept in culture for 63 days (TD63) to examine the effects of long-term culturing on the AD pathology manifestation and the expression of the P2X7R
Control and fAD, did not show major differences in the overall differentiation (TUBB3 and MAP2 expression), an increased astroglial differentiation was observed in the fAD cultures, which was demonstrated by Reverse transcription quantitative PCR (RT-qPCR) measurements (Figure 2A) and Western blot detection (GFAP expression) (Figures 2B,C)
Summary
Neuroinflammation is one of the major underlying pathologies of many neuropathological conditions, including Alzheimer’s disease (AD). This ATP serves as a damage-associated molecular pattern (DAMP) which is detected by microglial cells via their purinergic receptors From amongst both ionotropic (P2X) and metabotropic (P2Y) ATP-gated purinergic receptors (Huang et al, 2019), the P2X7R has attracted most attention due to its unique characteristics such as its low affinity to ATP (Surprenant et al, 1996) and the suggested involvement in a broad range of neurodegenerative diseases such as AD (Martin et al, 2019; Francistiová et al, 2020), epilepsy (Engel et al, 2012; Conte et al, 2020; Morgan et al, 2020), schizophrenia (Calovi et al, 2020), Huntington’s disease (Ollà et al, 2020) and many others. The formation of such a macropore in the cell membrane induces cell death and can be experimentally demonstrated by the uptake of large molecules, e.g., Yo-Pro-1 by the cells (Virginio et al, 1999; Faria et al, 2005)
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