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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N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine-and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo-and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1 ⌬NTD ) abrogating NR1 and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry.Excitatory neurotransmission in the mammalian brain is mainly mediated by ionotropic glutamate receptors (iGluRs). 4Based on pharmacological studies, iGluRs have been grouped into three distinct subfamilies: (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, kainate receptors, and N-methyl-D-aspartic acid (NMDA) receptors (1). All iGluR subunits share a common modular design of four distinct regions: (i) an extracellular N-terminal domain (NTD) of about 400 amino acids sharing homology with the bacterial leucine-, isoleucine-, and valine-binding protein, implicated to play a role in iGluR oligomerization and modulation, (ii) an extracellular S1S2 ligand binding domain sharing homology with the bacterial glutamine-binding protein that binds agonists in a Venus-flytrap like mechanism, (iii) a membrane-associated domain composed of four membrane segments forming the ion channel, and (iv) an intracellular C-terminal domain involved in linking the receptor to the membrane scaffold and signal transduction proteins (2).The NMDA subtype of iGluRs is an obligatory hetero-oligomeric membrane protein composed of homologous NR1, NR2, and/or NR3 subunits and plays a key role in brain development, synaptic plasticity, and memory formation (1). Cloning of NMDA receptor subunits revealed that the glycine-binding NR1 subunit is a single gene product occurring in eight splice variants (a-h), whereas the glutamate-binding NR2 and the glycine-binding NR3 subunits are encoded by four (NR2A-NR2D) and two (NR3A and -B) different genes...

Microglia, the innate immune cells of the CNS, play a pivotal role in brain injury and disease. Microglia are extremely motile; their highly ramified processes constantly survey the brain parenchyma, and they respond promptly to brain damage with targeted process movement toward the injury site. Microglia play a key role in brain development and function by pruning synapses during development, phagocytosing apoptotic newborn neurons, and regulating neuronal activity by direct microglia-neuron or indirect microglia-astrocyte-neuron interactions, which all depend on their process motility. This review highlights recent discoveries about microglial dynamics, focusing on the receptors, ion channels, and signaling pathways involved.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluRs) is a tetrameric protein composed of homologous NR1 and NR2 subunits, which require the binding of glycine and glutamate, respectively, for efficient channel gating. The extracellular N-terminal domains (NTDs) of iGluR subunits show sequence homology to the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) and have been implicated in iGluR assembly, trafficking, and function. Here, we investigated how deletion of the NR1-and NR2-NTDs affects the expression and function of NMDA receptors. Both proteolytic cleavage of the NR1-NTD from assembled NR1/ NR2 receptors and coexpression of the NTD-deleted NR1 subunit with wild-type or NTD-deleted NR2 subunits resulted in agonist-gated channels that closely resembled wild-type receptors. This indicates that the NTDs of both NMDA receptor subunits are not essential for receptor assembly and function. However, deletion of either the NR1 or the NR2 NTD eliminated high-affinity, allosteric inhibition of agonist-induced currents by Zn 2ϩ and ifenprodil, consistent with the idea that interdomain interactions between these domains are important for allosteric receptor modulation. Furthermore, by replacing the NR2A-NTD with the NR2B NTD, and vice versa, the different glycine affinities of NR1/NR2A and NR1/NR2B receptors were found to be determined by their respective NR2-NTDs. Together, these data show that the NTDs of both the NR1 and NR2 subunits determine allosteric inhibition and glycine potency but are not required for NMDA receptor assembly.

Essential microglial functions depend on membrane potential and ion channels.  Surveillance is regulated by THIK-1 K + channels, while directed process motility is controlled by purinergic signalling and Clchannels.  Enhanced K + efflux via THIK-1 and Kv channels is needed for NLRP3 inflammasome assembly.  Microglial ion channel and receptor expression in situ often differ from those in culture, which result in different morphological and functional states.

Neuronal gap junctional hemichannels, composed of pannexin-1 subunits, have been suggested to play a crucial role in epilepsy and brain ischaemia. After a few minutes of anoxia or ischaemia, neurons in brain slices show a rapid depolarization to ∼−20 mV, called the anoxic depolarization. Glutamate receptor blockers can prevent the anoxic depolarization, suggesting that it is produced by a cation influx through glutamate-gated channels. However, in isolated hippocampal pyramidal cells, simulated ischaemia evokes a large inward current and an increase in permeability to large molecules, mediated by the opening of pannexin-1 hemichannels. N-methyl-d-aspartate is also reported to open these hemichannels, suggesting that the activation of N-methyl-d-aspartate receptors, which occurs when glutamate is released in ischaemia, might cause the anoxic depolarization by evoking a secondary ion flux through pannexin-1 hemichannels. We tested the contribution of pannexin hemichannels to the anoxic depolarization in CA1 pyramidal cells in the more physiological environment of hippocampal slices. Three independent inhibitors of hemichannels—carbenoxolone, lanthanum and mefloquine—had no significant effect on the current generating the anoxic depolarization, while a cocktail of glutamate and gamma-aminobutyric acid class A receptor blockers abolished it. We conclude that pannexin hemichannels do not generate the large inward current that underlies the anoxic depolarization. Glutamate receptor channels remain the main candidate for generating the large inward current that produces the anoxic depolarization.