Carbon Monoxide, a Retrograde Messenger Generated in Postsynaptic Mushroom Body Neurons, Evokes Noncanonical Dopamine Release.

Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated. In Drosophila melanogaster brain explant preparations, DA is released specifically onto α3/α'3 compartments of mushroom body (MB) neurons that have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. Here we found that coincidentally activated MB neurons generate carbon monoxide (CO), which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in postsynaptic MB neurons, and CO-evoked DA release requires Ca2+ efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. We propose that DA neurons use two distinct modes of transmission to produce global and local DA signaling.SIGNIFICANCE STATEMENT Dopamine (DA) is needed for various higher brain functions, including memory formation. However, DA neurons form extensive synaptic connections, while memory formation requires highly specific and localized DA release. Here we identify a mechanism through which DA release from presynaptic terminals is controlled by postsynaptic activity. Postsynaptic neurons activated by cholinergic and glutamatergic inputs generate carbon monoxide, which acts as a retrograde messenger inducing presynaptic DA release. Released DA is required for memory-associated plasticity. Our work identifies a novel mechanism that restricts DA release to the specific postsynaptic sites that require DA during memory formation.

Detection and Removal of Endogenous Carbon Monoxide by Selective and Cell-Permeable Hemoprotein Model Complexes.

Carbon monoxide (CO) is produced in mammalian cells during heme metabolism and serves as an important signaling messenger. Here we report the bioactive properties of selective CO scavengers, hemoCD1 and its derivative R8-hemoCD1, which have the ability to detect and remove endogenous CO in cells. HemoCD1 is a supramolecular hemoprotein-model complex composed of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(II) and a per-O-methylated ß-cyclodextrin dimer having an pyridine linker. We demonstrate that hemoCD1 can be used effectively to quantify endogenous CO in cell lysates by a simple spectrophotometric method. The hemoCD1 assay detected ca. 260 pmol of CO in 106 hepatocytes, which was well-correlated with the amount of intracellular bilirubin, the final breakdown product of heme metabolism. We then covalently attached an octaarginine peptide to a maleimide-appended hemoCD1 to synthesize R8-hemoCD1, a cell-permeable CO scavenger. Indeed, R8-hemoCD1 was taken up by intact cells and captured intracellular CO with high efficiency. Moreover, we revealed that removal of endogenous CO by R8-hemoCD1 in cultured macrophages led to a significant increase (ca. 2.5-fold) in reactive oxygen species production and exacerbation of inflammation after challenge with lipopolysaccharide. Thus, R8-hemoCD1 represents a powerful expedient for exploring specific and still unidentified biological functions of CO in cells.

Iron(II)porphyrin-Cyclodextrin Supramolecular Complex as a Carbon Monoxide-Depleting Agent in Living Organisms.

The specific intermolecular interaction between an anionic tetraarylporphyrin and per-O-methylated ß-cyclodextrin (TMe-ß-CD) paved the way to produce a functional supramolecule that works as a strong carbon monoxide (CO)-depleting agent in living organisms. The supramolecular complex, hemoCD, that is composed of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(II) and a TMe-ß-CD dimer linked by a pyridine linker, captured internal CO from carboxyhemoglobin during its circulation in the blood of animals. HemoCD thus produced the pseudo-knockdown (loss-of-functional) state of endogenous CO in the animals. This unique property led us to investigate the biological function of endogenous CO as a gaseous signal mediator in living systems. In this paper, we introduce our recent study on the hemoCD complex as a biological CO-depleting agent.

Feedback Response to Selective Depletion of Endogenous Carbon Monoxide in the Blood.

The physiological roles of endogenous carbon monoxide (CO) have not been fully understood because of the difficulty in preparing a loss-of-function phenotype of this molecule. Here, we have utilized in vivo CO receptors, hemoCDs, which are the supramolecular 1:1 inclusion complexes of meso-tetrakis(4-sulfonatophenyl)porphinatoiron(II) with per-O-methylated ß-cyclodextrin dimers. Three types of hemoCDs (hemoCD1, hemoCD2, and hemoCD3) that exhibit different CO-affinities have been tested as CO-depleting agents in vivo. Intraperitoneally administered hemoCD bound endogenous CO within the murine circulation, and was excreted in the urine along with CO in an affinity-dependent manner. The sufficient administration of hemoCD that has higher CO-affinity than hemoglobin (Hb) produced a pseudoknockdown state of CO in the mouse in which heme oxygenase-1 (HO-1) was markedly induced in the liver, causing the acceleration of endogenous CO production to maintain constant CO-Hb levels in the blood. The contents of free hemin and bilirubin in the blood plasma of the treated mice significantly increased upon removal of endogenous CO by hemoCD. Thus, a homeostatic feedback model for the CO/HO-1 system was proposed as follows: HemoCD primarily removes CO from cell-free CO-Hb. The resulting oxy-Hb is quickly oxidized to met-Hb by oxidant(s) such as hydrogen peroxide in the blood plasma. The met-Hb readily releases free hemin that directly induces HO-1 in the liver, which metabolizes the hemin into iron, biliverdin, and CO. The newly produced CO binds to ferrous Hb to form CO-Hb as an oxidation-resistant state. Overall, the present system revealed the regulatory role of CO for maintaining the ferrous/ferric balance of Hb in the blood.

Circadian clock disruption by selective removal of endogenous carbon monoxide.

Circadian rhythms are regulated by transcription-translation feedback loops (TTFL) of clock genes. Previous studies have demonstrated that core transcriptional factors, NPAS2 and CLOCK, in the TTFL can reversibly bind carbon monoxide (CO) in vitro. However, little is known about whether endogenous CO, which is continuously produced during a heme metabolic process, is involved in the circadian system. Here we show that selective removal of endogenous CO in mice considerably disrupts rhythmic expression of the clock genes. A highly selective CO scavenger, hemoCD1, which is a supramolecular complex of an iron(II)porphyrin with a per-O-methyl-ß-cyclodextrin dimer, was used to remove endogenous CO in mice. Intraperitoneal administration of hemoCD1 to mice immediately reduced the amount of internal CO. The removal of CO promoted the bindings of NPAS2 and CLOCK to DNA (E-box) in the murine liver, resulting in up-regulation of the E-box-controlled clock genes (Per1, Per2, Cry1, Cry2, and Rev-erbα). Within 3 h after the administration, most hemoCD1 in mice was excreted in the urine, and heme oxygenase-1 (HO-1) was gradually induced in the liver. Increased endogenous CO production due to the overexpression of HO-1 caused dissociation of NPAS2 and CLOCK from E-box, which in turn induced down-regulation of the clock genes. The down-regulation continued over 12 h even after the internal CO level recovered to normal. The late down-regulation was ascribed to an inflammatory response caused by the endogenous CO reduction. The CO pseudo-knockdown experiments provided the clear evidence that endogenous CO contributes to regulation in the mammalian circadian clock.

Surface modification of a polyvinyl alcohol sponge with functionalized boronic acids to develop porous materials for multicolor emission, chemical sensing and 3D cell culture.

Surface modification of a polyvinyl alcohol sponge with functionalized boronic acids led to the formation of porous materials applicable for multicolor emission, chemical sensing and 3D cell culture.