Abstract:ADP-ribosylation of the P2X7k splice variant on mouse T cells by Ecto-ADPribosyltransferase ARTC2.2 in response to its substrate extracellular nicotinamide adenine dinucleotide (NAD + ) triggers cell death. Since NAD + is released as a danger signal during tissue damage, this NAD + -induced cell death (NICD) may impact the survival of other cell populations co-expressing P2X7 and of one of the ARTC2 isoforms (ARTC2.1, ARTC2.2). NICD of brain-resident, non-T cell populations has only been rudimentarily investig… Show more
“…Isolation of primary cells. Isolation of primary cells was described before 50 . For the isolation of brain microglia, mice were sacrificed under anesthesia by cervical dislocation.…”
Section: Sds-page and Western Blot Analysismentioning
The ATP-gated P2X7 receptor is highly expressed in microglia and has been involved in diverse brain diseases. P2X7 effects were also described in neurons and astrocytes but its localisation and function in these cell types has been challenging to demonstrate in situ. BAC transgenic mouse lines have greatly advanced neuroscience research and two BAC-transgenic P2X7 reporter mouse models exist in which either a soluble EGFP (sEGFP) or an EGFP-tagged P2X7 receptor (P2X7-EGFP) is expressed under the control of a BAC-derived P2rx7 promoter. Here we evaluate both mouse models and find striking differences in both P2X expression levels and EGFP reporter expression patterns. Most remarkably, the sEGFP model overexpresses a P2X4 passenger gene and sEGFP shows clear neuronal localisation but appears to be absent in microglia. Preliminary functional analysis in a status epilepticus model suggests functional consequences of the observed P2X receptor overexpression. In summary, an aberrant EGFP reporter pattern and possible effects of P2X4 and/or P2X7 protein overexpression need to be considered when working with this model. We further discuss reasons for the observed differences and possible caveats in BAC transgenic approaches.
“…Isolation of primary cells. Isolation of primary cells was described before 50 . For the isolation of brain microglia, mice were sacrificed under anesthesia by cervical dislocation.…”
Section: Sds-page and Western Blot Analysismentioning
The ATP-gated P2X7 receptor is highly expressed in microglia and has been involved in diverse brain diseases. P2X7 effects were also described in neurons and astrocytes but its localisation and function in these cell types has been challenging to demonstrate in situ. BAC transgenic mouse lines have greatly advanced neuroscience research and two BAC-transgenic P2X7 reporter mouse models exist in which either a soluble EGFP (sEGFP) or an EGFP-tagged P2X7 receptor (P2X7-EGFP) is expressed under the control of a BAC-derived P2rx7 promoter. Here we evaluate both mouse models and find striking differences in both P2X expression levels and EGFP reporter expression patterns. Most remarkably, the sEGFP model overexpresses a P2X4 passenger gene and sEGFP shows clear neuronal localisation but appears to be absent in microglia. Preliminary functional analysis in a status epilepticus model suggests functional consequences of the observed P2X receptor overexpression. In summary, an aberrant EGFP reporter pattern and possible effects of P2X4 and/or P2X7 protein overexpression need to be considered when working with this model. We further discuss reasons for the observed differences and possible caveats in BAC transgenic approaches.
“…GLAST1 is a known cell surface marker expressed on microglia and astrocytes 50 . To differentiate between the microglia and astrocytes in all of the samples, flow cytometry analyses were done, and microglia were identified as CD11b + GLAST1 − cells and astrocytes as CD11b − GLAST1 + , as previously explained 51 . Fig.…”
AbstractβA3/A1-crystallin, a lens protein that is also expressed in astrocytes, is produced as βA3 and βA1-crystallin isoforms by leaky ribosomal scanning. In a previous human proteome high-throughput array, we found that βA3/A1-crystallin interacts with protein tyrosine phosphatase 1B (PTP1B), a key regulator of glucose metabolism. This prompted us to explore possible roles of βA3/A1-crystallin in metabolism of retinal astrocytes. We found that βA1-crystallin acts as an uncompetitive inhibitor of PTP1B, but βA3-crystallin does not. Loss of βA1-crystallin in astrocytes triggers metabolic abnormalities and inflammation. In CRISPR/cas9 gene-edited βA1-knockdown (KD) mice, but not in βA3-knockout (KO) mice, the streptozotocin (STZ)-induced diabetic retinopathy (DR)-like phenotype is exacerbated. Here, we have identified βA1-crystallin as a regulator of PTP1B; loss of this regulation may be a new mechanism by which astrocytes contribute to DR. Interestingly, proliferative diabetic retinopathy (PDR) patients showed reduced βA1-crystallin and higher levels of PTP1B in the vitreous humor.
“…Recently, Rissiek et al (2020) reported that the dominant P2X7 receptor in mouse astrocytes in culture is P2X7a, a splice variant that is much less sensitive to NAD + /ARTC2‐mediated ADP‐ribosylation (Rissiek et al, 2020). In the present study, however, we showed that P2X7 receptor‐mediated responses were dramatically increased by NAD + in astrocytes.…”
We previously showed that noninvasive mild ischemia (preconditioning; PC) induced ischemic tolerance by upregulation of P2X7 receptors in astrocytes via a hypoxia inducible factor‐1α (HIF‐1α)‐dependent mechanism. The P2X7 receptor is known as a low‐sensitivity P2 receptor that requires a high extracellular ATP (eATP) concentration for activation. PC increased the eATP level but was not sufficient to activate P2X7 receptors. Here, we show that astrocytes possess an elaborate mechanism for activation of P2X7 receptors, thus contributing to ischemic tolerance. Nicotinamide adenine dinucleotide (NAD+) was shown to increase the sensitivity of P2X7 receptors to eATP via ecto‐ADP‐ribosyltransferase 2 (ARTC2)‐catalyzed ADP‐ribosylation in peripheral immune cells. Although ARTC2‐positive signals were mostly absent in the naïve brain, they were selectively increased in astrocytes by PC. The spatiotemporal pattern of PC‐evoked ARTC2 was well associated with that of P2X7 receptors. In the in vitro experiments, NAD+ increased the sensitivity of P2X7 receptors to ATP, and at higher concentrations, NAD+ itself activated P2X7 receptors without eATP in cultured astrocytes. In the in vivo experiments using middle cerebral artery occlusion model mice, the PC‐evoked increase in HIF‐1α in astrocytes was abolished by the ARTC2 inhibitor S + 16a. S + 16a also abolished PC‐evoked ischemic tolerance. Taken together, the results suggested that P2X7 receptors can be sensitized to ATP by NAD+/ARTC2‐catalyzed ADP‐ribosylation, which allows astrocytes to drive P2X7 receptor‐mediated ischemic tolerance even though PC only slightly increases the amount of eATP.
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