Rissiek, B. | bioRxiv Science

bioRxiv2020

A P2rx7 passenger mutation affects the vitality and function of immune cells in P2X4ko and other transgenic mice

Among laboratory mouse strains many genes are differentially expressed in the same cell population. As consequence, gene targeting in 129-derived embryonic stem cells (ESCs) and backcrossing the modified mice onto the C57BL/6 (B6) background can introduce passenger mutations in the close proximity of the targeted gene. Here, we demonstrate that several 129-originating transgenic mice in which P2rx7-neighboring genes were targeted carry a P2rx7 passenger mutation that affects the vitality and function of T cells. By the example of P2rx4tm1Rass we demonstrate that CD4+ and CD8+ T cells derived from these mice express higher levels of P2X7 when compared to corresponding cell populations in B6-WT mice. The increased T cell sensitivity towards the P2X7 activators adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+) rendered these cells more vulnerable towards NAD-induced cell death (NICD) compared to their B6-WT counterparts. The enhanced NICD sensitivity significantly affected the outcome of functional assays e.g. cytokine production and cell migration. For P2rx4tm1Rass, we demonstrate that the expression of P2X7 is diminished in several innate immune cell populations, possibly as a side effect of P2rx4 targeting, and independent of the P2rx7 passenger mutation. These results need to be considered when working with P2rx4tm1Rass mice or other 129-based transgenic strains that target P2rx7 neighboring genes and might have implications for other mouse models.

immunology↗

bioRxiv2019

BRAIN-DERIVED EXTRACELLULAR VESICLES ARE HIGHLY ENRICHED IN THE PRION PROTEIN AND ITS C1 FRAGMENT: RELEVANCE FOR CELLULAR UPTAKE AND IMPLICATIONS IN STROKE

Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischemic region. The fate of neurons in the surrounding penumbra depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions. In the present study, we characterize the EVs in mouse brain under physiological conditions and 24h after induction of transient ischemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs) whereas after ischemia, the main EV population originates from astrocytes. Moreover, sEVs presented high amounts of the prion protein (PrP) which were increased after stroke. Conspicuously, sEVs were particularly enriched in a truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or containing PrP (WT). We show that PrP-KO-EVs are rapidly taken up by neurons and colocalize with lysosomes. Although eventually WT-EVs are also found in lysosomes, the amount taken up by neurons is significantly higher for PrP-KO-EVs. Likewise, microglia and astrocytes were also engulfing PrP-KO-sEVs more efficiently than WT-sEVs. Our results provide information on the relative contribution of brain cell types to the sEV pool in mice and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to their cellular uptake.

neuroscience↗