Cerebral blood flow is reduced early in the onset of Alzheimer’s disease (AD). Because most of the vascular resistance within the brain is in capillaries, this could reflect dysfunction of contractile pericytes on capillary walls. We used live and rapidly fixed biopsied human tissue to establish disease relevance, and rodent experiments to define mechanism. We found that in humans with cognitive decline, amyloid β (Aβ) constricts brain capillaries at pericyte locations. This was caused by Aβ generating reactive oxygen species, which evoked the release of endothelin-1 (ET) that activated pericyte ETA receptors. Capillary, but not arteriole, constriction also occurred in vivo in a mouse model of AD. Thus, inhibiting the capillary constriction caused by Aβ could potentially reduce energy lack and neurodegeneration in AD.

SummaryMicroglia exhibit two modes of motility: they constantly extend and retract their processes to survey the brain, but they also send out targeted processes to envelop sites of tissue damage. We now show that these motility modes differ mechanistically. We identify the two-pore domain channel THIK-1 as the main K+ channel expressed in microglia in situ. THIK-1 is tonically active, and its activity is potentiated by P2Y12 receptors. Inhibiting THIK-1 function pharmacologically or by gene knockout depolarizes microglia, which decreases microglial ramification and thus reduces surveillance, whereas blocking P2Y12 receptors does not affect membrane potential, ramification, or surveillance. In contrast, process outgrowth to damaged tissue requires P2Y12 receptor activation but is unaffected by blocking THIK-1. Block of THIK-1 function also inhibits release of the pro-inflammatory cytokine interleukin-1β from activated microglia, consistent with K+ loss being needed for inflammasome assembly. Thus, microglial immune surveillance and cytokine release require THIK-1 channel activity.

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Microglia, the brain's innate immune cells, have highly motile processes which constantly survey the brain to detect infection, remove dying cells and prune synapses during brain development. ATP released by tissue damage is known to attract microglial processes, but it is controversial whether an ambient level of ATP is needed to promote constant microglial surveillance in the normal brain. Applying the ATPase apyrase, an enzyme which hydrolyses ATP and ADP, reduces microglial process ramification and surveillance, suggesting that ambient ATP/ADP maintains microglial surveillance. However, attempting to raise the level of ATP/ADP by blocking the endogenous ecto-ATPase (termed NTPDase1/CD39), which also hydrolyses ATP/ADP, does not affect the cells' ramification or surveillance, nor their membrane currents which respond to even small rises of extracellular [ATP] or [ADP] with the activation of K + channels. This indicates a lack of detectable ambient ATP/ADP and ecto-ATPase activity, contradicting the results with apyrase. We resolve this contradiction by demonstrating that contamination of commercially-available apyrase by a high K + concentration reduces ramification and surveillance by depolarising microglia.Exposure to the same K + concentration (without apyrase added) reduced ramification and surveillance as with apyrase. Dialysis of apyrase to remove K + retained its ATP-hydrolysing activity but abolished the microglial depolarisation and decrease of ramification produced by the undialysed enzyme. Thus, applying apyrase affects microglia by an action independent of ATP, and no ambient purinergic signalling is required to maintain microglial ramification and surveillance. These results also have implications for hundreds of prior studies that employed apyrase to hydrolyse ATP/ADP. Key words: microglia, ATP, surveillance, apyraseSignificance statement ATP mediates interactions between cells in many tissues, but is particularly important for microglia, the brain's immune cells, which constantly survey the brain to detect infection and to regulate the brain's wiring during development. It is controversial whether the ceaseless movement of microglia is driven by ATP release from brain cells. We show that an enzyme (apyrase) widely used to manipulate ATP levels is contaminated with K + ions which inhibit microglial surveillance, and that no ATP release is needed to drive microglial process movement. Thus, all conclusions about a role of ATP in signalling based on applying apyrase need re-examining, and brain immune surveillance is not regulated by ATP release. \body IntroductionATP-mediated signalling is present in many tissues, but is particularly important for microglia, the brain's innate immune cells (1). Microglia constantly survey the brain by extending and retracting their processes to sense their environment (2) but also, in the case of sudden brain damage, promptly send out processes to quickly target and enclose the site of injury (3). The latter response is mediated by ATP released from the...

Microglia sense their environment using an array of membrane receptors. While P2Y12 receptors are known to play a key role in targeting directed motility of microglial processes to sites of damage where ATP/ADP is released, little is known about the role of P2Y13, which transcriptome data suggest is the second most expressed neurotransmitter receptor in microglia. We show that, in patch‐clamp recordings in acute brain slices from mice lacking P2Y13 receptors, the THIK‐1 K+ current density evoked by ADP activating P2Y12 receptors was increased by ~50%. This increase suggested that the P2Y12‐dependent chemotaxis response should be potentiated; however, the time needed for P2Y12‐mediated convergence of microglial processes onto an ADP‐filled pipette or to a laser ablation was longer in the P2Y13 KO. Anatomical analysis showed that the density of microglia was unchanged, but that they were less ramified with a shorter process length in the P2Y13 KO. Thus, chemotactic processes had to grow further and so arrived later at the target, and brain surveillance was reduced by ~30% in the knock‐out. Blocking P2Y12 receptors in brain slices from P2Y13 KO mice did not affect surveillance, demonstrating that tonic activation of these high‐affinity receptors is not needed for surveillance. Strikingly, baseline interleukin‐1β release was increased fivefold while release evoked by LPS and ATP was not affected in the P2Y13 KO, and microglia in intact P2Y13 KO brains were not detectably activated. Thus, P2Y13 receptors play a role different from that of their close relative P2Y12 in regulating microglial morphology and function.