A critical role of TRPM2 channel in Aß42 -induced microglial activation and generation of tumor necrosis factor-α.

Amyloid ß (Aß)-induced neuroinflammation plays an important part in Alzheimer's disease (AD). Emerging evidence supports a role for the transient receptor potential melastatin-related 2 (TRPM2) channel in Aß-induced neuroinflammation, but how Aß induces TRPM2 channel activation and this relates to neuroinflammation remained poorly understood. We investigated the mechanisms by which Aß42 activates the TRPM2 channel in microglial cells and the relationships to microglial activation and generation of tumor necrosis factor-α (TNF-α), a key cytokine implicated in AD. Exposure to 10-300 nM Aß42 induced concentration-dependent microglial activation and generation of TNF-α that were ablated by genetically deleting (TRPM2 knockout ;TRPM2-KO) or pharmacologically inhibiting the TRPM2 channel, revealing a critical role of this channel in Aß42 -induced microglial activation and generation of TNF-α. Mechanistically, Aß42 activated the TRPM2 channel via stimulating generation of reactive oxygen species (ROS) and activation of poly(ADPR) polymerase-1 (PARP-1). Aß42 -induced generation of ROS and activation of PARP-1 and TRPM2 channel were suppressed by inhibiting protein kinase C (PKC) and NADPH oxidases (NOX). Aß42 -induced activation of PARP-1 and TRPM2 channel was also reduced by inhibiting PYK2 and MEK/ERK. Aß42 -induced activation of PARP-1 was attenuated by TRPM2-KO and moreover, the remaining PARP-1 activity was eliminated by inhibiting PKC and NOX, but not PYK2 and MEK/ERK. Collectively, our results suggest that PKC/NOX-mediated generation of ROS and subsequent activation of PARP-1 play a role in Aß42 -induced TRPM2 channel activation and TRPM2-dependent activation of the PYK2/MEK/ERK signalling pathway acts as a positive feedback to further facilitate activation of PARP-1 and TRPM2 channel. These findings provide novel insights into the mechanisms underlying Aß-induced AD-related neuroinflammation.

Signalling mechanisms mediating Zn2+-induced TRPM2 channel activation and cell death in microglial cells.

Excessive Zn2+ causes brain damage via promoting ROS generation. Here we investigated the role of ROS-sensitive TRPM2 channel in H2O2/Zn2+-induced Ca2+ signalling and cell death in microglial cells. H2O2/Zn2+ induced concentration-dependent increases in cytosolic Ca2+ concentration ([Ca2+]c), which was inhibited by PJ34, a PARP inhibitor, and abolished by TRPM2 knockout (TRPM2-KO). Pathological concentrations of H2O2/Zn2+ induced substantial cell death that was inhibited by PJ34 and DPQ, PARP inhibitors, 2-APB, a TRPM2 channel inhibitor, and prevented by TRPM2-KO. Further analysis indicate that Zn2+ induced ROS production, PARP-1 stimulation, increase in the [Ca2+]c and cell death, all of which were suppressed by chelerythrine, a protein kinase C inhibitor, DPI, a NADPH-dependent oxidase (NOX) inhibitor, GKT137831, a NOX1/4 inhibitor, and Phox-I2, a NOX2 inhibitor. Furthermore, Zn2+-induced PARP-1 stimulation, increase in the [Ca2+]c and cell death were inhibited by PF431396, a Ca2+-sensitive PYK2 inhibitor, and U0126, a MEK/ERK inhibitor. Taken together, our study shows PKC/NOX-mediated ROS generation and PARP-1 activation as an important mechanism in Zn2+-induced TRPM2 channel activation and, TRPM2-mediated increase in the [Ca2+]c to trigger the PYK2/MEK/ERK signalling pathway as a positive feedback mechanism that amplifies the TRPM2 channel activation. Activation of these TRPM2-depenent signalling mechanisms ultimately drives Zn2+-induced Ca2+ overloading and cell death.

Purinergic and Store-Operated Ca(2+) Signaling Mechanisms in Mesenchymal Stem Cells and Their Roles in ATP-Induced Stimulation of Cell Migration.

ATP is an extrinsic signal that can induce an increase in the cytosolic Ca(2+) level ([Ca(2+) ]c ) in mesenchymal stem cells (MSCs). However, the cognate intrinsic mechanisms underlying ATP-induced Ca(2+) signaling in MSCs is still contentious, and their importance in MSC migration remains unknown. In this study, we investigated the molecular mechanisms underlying ATP-induced Ca(2+) signaling and their roles in the regulation of cell migration in human dental pulp MSCs (hDP-MSCs). RT-PCR analysis of mRNA transcripts and interrogation of agonist-induced increases in the [Ca(2+) ]c support that P2X7, P2Y1 , and P2Y11 receptors participate in ATP-induced Ca(2+) signaling. In addition, following P2Y receptor activation, Ca(2+) release-activated Ca(2+) Orai1/Stim1 channel as a downstream mechanism also plays a significant role in ATP-induced Ca(2+) signaling. ATP concentration-dependently stimulates hDP-MSC migration. Pharmacological and genetic interventions of the expression or function of the P2X7, P2Y1 and P2Y11 receptors, and Orai1/Stim1 channel support critical involvement of these Ca(2+) signaling mechanisms in ATP-induced stimulation of hDP-MSC migration. Taken together, this study provide evidence to show that purinergic P2X7, P2Y1 , and P2Y11 receptors and store-operated Orai1/Stim1 channel represent important molecular mechanisms responsible for ATP-induced Ca(2+) signaling in hDP-MSCs and activation of these mechanisms stimulates hDP-MSC migration. Such information is useful in building a mechanistic understanding of MSC homing in tissue homeostasis and developing more efficient MSC-based therapeutic applications. Stem Cells 2016;34:2102-2114.

Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Functional properties of five Dictyostelium discoideum P2X receptors.

The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

Role of P2X4 receptors in synaptic strengthening in mouse CA1 hippocampal neurons.

P2X4 receptors are calcium-permeable cation channels gated by extracellular ATP. They are found close to subsynaptic sites on hippocampal CA1 neurons. We compared features of synaptic strengthening between wild-type and P2X4 knockout mice (21-26 days old). Potentiation evoked by a tetanic presynaptic stimulus (100 Hz, 1 s) paired with postsynaptic depolarization was less in P2X4(-/-) mice than in wild-type mice (230 vs. 50% potentiation). Paired-pulse ratios and the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) were not different between wild-type and knockout mice. Prior hyperpolarization (ten 3 s pulses to -120 mV at 0.17 Hz) potentiated the amplitude of spontaneous EPSCs in wild-type mice, but not in P2X4(-/-) mice; this potentiation was not affected by nifedipine, but was abolished by 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA) in the recording pipette. The amplitude of N-methyl-d-aspartate EPSCs (in 6-cyano-7-nitroquinoxaline-2,3-dione, 10 or 30 µm, at -100 mV) facilitated during 20 min recording in magnesium-free solution. In wild-type mice, this facilitation of the N-methyl-d-aspartate EPSC was reduced by about 50% by intracellular BAPTA (10 mM), ifenprodil (3 µm) or 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 µm). In P2X4(-/-) mice, the facilitation was much less, and was unaffected by intracellular BAPTA, ifenprodil (3 µm) or mitogen-activated protein (MAP) kinase inhibitor 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (5 µm). This suggests that the absence of P2X4 receptors limits the incorporation of NR2B subunits into synaptic N-methyl-d-aspartate receptors.

Ectodomain lysines and suramin block of P2X1 receptors.

P2X(1) receptors belong to a family of cation channels gated by extracellular ATP; they are found inter alia in smooth muscle, platelets, and immune cells. Suramin has been widely used as an antagonist at P2X receptors, and its analog 4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino))] tetrakis-benzene-1,3-disulfonic acid (NF449) is selective for the P2X(1) subtype. Human and mouse P2X(1) receptors were expressed in human embryonic kidney cells, and membrane currents evoked by ATP were recorded. ATP (10 nm to 100 microm) was applied only once to each cell, to avoid the profound desensitization exhibited by P2X(1) receptors. Suramin (10 microm) and NF449 (3-300 nM) effectively blocked the human receptor. Suramin had little effect on the mouse receptor. Suramin and NF449 are polysulfonates, with six and eight negative charges, respectively. We hypothesized that species differences might result from differences in positive residues presented by the large receptor ectodomain. Four lysines in the human sequence (Lys(111), Lys(127), Lys(138), and Lys(148)) were changed individually and together to their counterparts in the mouse sequence. The substitution K138E, either alone or together with K111Q, K127Q, and K148N, reduced the sensitivity to block by both suramin and NF449. Conversely, when lysine was introduced into the mouse receptor, the sensitivity to block by suramin and NF449 was much increased for E138K, but not for Q111K, Q127K, or N148K. The results explain the marked species difference in antagonist sensitivity and identify an ectodomain lysine residue that plays a key role in the binding of both suramin and NF449 to P2X(1) receptors.

Altered hippocampal synaptic potentiation in P2X4 knock-out mice.

P2X4 purinergic receptors are calcium-permeable, ATP-activated ion channels. In the CA1 area of the hippocampus, they are located at the subsynaptic membrane somewhat peripherally to AMPA receptors. The possible role of P2X4 receptors has been difficult to elucidate because of the lack of selective antagonists. Here we report the generation of a P2X4 receptor knock-out mouse and show that long-term potentiation (LTP) at Schaffer collateral synapses is reduced relative to that in wild-type mice. Ivermectin, which selectively potentiates currents at P2X4, was found to increase LTP in wild-type mice but had no effect in P2X4 knock-out mice. We suggest that calcium entry through subsynaptic P2X4 receptors during high-frequency stimulation contributes to synaptic strengthening.

Reanalysis of P2X7 receptor expression in rodent brain.

P2X receptors are cationic-selective ion channels gated by extracellular ATP. There are seven subunits (P2X1-7), the first six of which are expressed throughout the peripheral and central nervous systems. P2X7 receptors are rapidly upregulated and activated as a result of inflammatory stimuli in immune cells, where they act not only as cationic channels but uniquely couple with rapid release of proinflammatory cytokines, cytoskeletal rearrangements, and apoptosis or necrotic cell death. The P2X7 receptor has been termed the cytolytic non-neuronal P2X receptor because it had not been detected in neurons until recently when it has been immunolocalized to several brain regions, particularly the hippocampus, and has been suggested to be involved in presynaptic modulation of transmitter release. Because its expression in brain neurons may have substantial functional implications, we have performed detailed immunocytochemical, immunoblot, and immunoprecipitation studies on brain and non-neuronal tissue using all currently available antibodies. We first examined rats, but staining patterns were inconsistent among antibodies; we therefore studied mice for which there are two P2X7 knock-out mice constructs available, one expressing the LacZ transgene. We found that P2X7 receptor protein is strongly and reliably detected in the submandibular gland and lung of wild-type mice but not in either of the P2X7-/- mice. However, we failed to find evidence for P2X7 receptor protein in hippocampal neurons or their input-output projections. Either the P2X7 protein in the hippocampus is below the limits of detection by the currently available methods or it is not present.