Contribution of astrocytic histamine N-methyltransferase to histamine clearance and brain function in mice.

Brain histamine acts as a neurotransmitter in the regulation of various brain activities. Previous studies have shown that histamine N-methyltransferase (HNMT), a histamine-metabolizing enzyme, controls brain histamine concentration and brain function. However, the relative contribution of astrocytic or neuronal HNMT to the regulation of the histaminergic system is still inconclusive. Here, we phenotyped astrocytes-specific HNMT knockout (cKO) mice to clarify the involvement of astrocytic HNMT in histamine clearance and brain function. First, we performed histological examinations using HNMT reporter mice and showed a wide distribution of HNMT in the brain and astrocytic HNMT expression. Then, we created cKO mice by Cre-loxP system and confirmed that HNMT expression in cKO primary astrocytes was robustly decreased. Although total HNMT level in the cortex was not substantially different between control and cKO brains, histamine concentration after histamine release was elevated in cKO cortex. In behavioral tests, impaired motor coordination and lower locomotor activity were observed in the cKO mice. However, anxiety-like behaviors, depression-like behaviors, and memory functions were not altered by astrocytic HNMT disruption. Although sleep analysis demonstrated that the quantity of wakefulness and sleep did not change, the increased power density of delta frequency during wakefulness indicated lower cortical activation in cKO mice. These results demonstrate that astrocytic HNMT contributes to histamine clearance after histamine release in the cortex and plays a role in the regulation of motor coordination, locomotor activity, and vigilance state.

Heparan sulfate promotes differentiation of white adipocytes to maintain insulin sensitivity and glucose homeostasis.

Heparan sulfate (HS), a highly sulfated linear polysaccharide, is involved in diverse biological functions in various tissues. Although previous studies have suggested a possible contribution of HS to the differentiation of white adipocytes, there has been no direct evidence supporting this. Here, we inhibited the synthesis of HS chains in 3T3-L1 cells using CRISPR-Cas9 technology, resulting in impaired differentiation of adipocytes with attenuated bone morphogenetic protein 4 (BMP4)-fibroblast growth factor 1 (FGF1) signaling pathways. HS reduction resulted in reduced glucose uptake and decreased insulin-dependent intracellular signaling. We then made heterozygous mutant mice for the Ext1 gene, which encodes an enzyme essential for the HS biosynthesis, specifically in the visceral white adipose tissue (Fabp4-Cre+::Ext1flox/WT mice, hereafter called Ext1Δ/WT) to confirm the importance of HS in vivo. The expression levels of transcription factors that control adipocyte differentiation, such as peroxisome proliferator-activated receptor gamma, were reduced in Ext1Δ/WT adipocytes, which contained smaller, unilocular lipid droplets, reduced levels of enzymes involved in lipid synthesis, and altered expression of BMP4-FGF1 signaling molecules. Furthermore, we examined the impact of HS reduction in visceral white adipose tissue on systemic glucose homeostasis. We observed that Ext1Δ/WT mice showed glucose intolerance because of insulin resistance. Our results demonstrate that HS plays a crucial role in the differentiation of white adipocytes through BMP4-FGF1 signaling pathways, thereby contributing to insulin sensitivity and glucose homeostasis.

Heparan sulfate controls skeletal muscle differentiation and motor functions.

BACKGROUND: Heparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear. METHODS: We examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system. RESULTS: CRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl2 injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells. SIGNIFICANCE: These results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.

Histamine H1 receptor on astrocytes and neurons controls distinct aspects of mouse behaviour.

Histamine is an important neurotransmitter that contributes to various processes, including the sleep-wake cycle, learning, memory, and stress responses. Its actions are mediated through histamine H1-H4 receptors. Gene knockout and pharmacological studies have revealed the importance of H1 receptors in learning and memory, regulation of aggression, and wakefulness. H1 receptors are abundantly expressed on neurons and astrocytes. However, to date, studies selectively investigating the roles of neuronal and astrocytic H1 receptors in behaviour are lacking. We generated novel astrocyte- and neuron-specific conditional knockout (cKO) mice to address this gap in knowledge. cKO mice showed cell-specific reduction of H1 receptor gene expression. Behavioural assessment revealed significant changes and highlighted the importance of H1 receptors on both astrocytes and neurons. H1 receptors on both cell types played a significant role in anxiety. Astrocytic H1 receptors were involved in regulating aggressive behaviour, circadian rhythms, and quality of wakefulness, but not sleep behaviour. Our results emphasise the roles of neuronal H1 receptors in recognition memory. In conclusion, this study highlights the novel roles of H1 receptors on astrocytes and neurons in various brain functions.

Brain histamine H1 receptor occupancy after oral administration of desloratadine and loratadine.

Some histamine H1 receptor (H1R) antagonists induce adverse sedative reactions caused by blockade of histamine transmission in the brain. Desloratadine is a second-generation antihistamine for treatment of allergic disorders. Its binding to brain H1Rs, which is the basis of sedative property of antihistamines, has not been examined previously in the human brain by positron emission tomography (PET). We examined brain H1R binding potential ratio (BPR), H1R occupancy (H1RO), and subjective sleepiness after oral desloratadine administration in comparison to loratadine. Eight healthy male volunteers underwent PET imaging with [11C]-doxepin, a PET tracer for H1Rs, after a single oral administration of desloratadine (5 mg), loratadine (10 mg), or placebo in a double-blind crossover study. BPR and H1RO in the cerebral cortex were calculated, and plasma concentrations of loratadine and desloratadine were measured. Subjective sleepiness was quantified by the Line Analogue Rating Scale (LARS) and the Stanford Sleepiness Scale (SSS). BPR was significantly lower after loratadine administration than after placebo (0.504 ± 0.074 vs 0.584 ± 0.059 [mean ± SD], P < 0.05), but BPR after desloratadine administration was not significantly different from BPR after placebo (0.546 ± 0.084 vs 0.584 ± 0.059, P = 0.250). The plasma concentration of loratadine was negatively correlated with BPR in subjects receiving loratadine, but that of desloratadine was not correlated with BPR. Brain H1ROs after desloratadine and loratadine administration were 6.47 ± 10.5% and 13.8 ± 7.00%, respectively (P = 0.103). Subjective sleepiness did not significantly differ among subjects receiving the two antihistamines and placebo. At therapeutic doses, desloratadine did not bind significantly to brain H1Rs and did not induce any significant sedation.

Histamine elicits glutamate release from cultured astrocytes.

Astrocytes play key roles in regulating brain homeostasis and neuronal activity. This is, in part, accomplished by the ability of neurotransmitters in the synaptic cleft to bind astrocyte membrane receptors, activating signalling cascades that regulate concentration of intracellular Ca2+ ([Ca2+]i) and gliotransmitter release, including ATP and glutamate. Gliotransmitters contribute to dendrite formation and synaptic plasticity, and in some cases, exacerbate neurodegeneration. The neurotransmitter histamine participates in several physiological processes, such as the sleep-wake cycle and learning and memory. Previous studies have demonstrated the expression of histamine receptors on astrocytes, but until now, only a few studies have examined the effects of histamine on astrocyte intracellular signalling and gliotransmitter release. Here, we used the human astrocytoma cell line 1321N1 to study the role of histamine in astrocyte intracellular signalling and gliotransmitter release. We found that histamine activated astrocyte signalling through histamine H1 and H2 receptors, leading to distinct cellular responses. Activation of histamine H1 receptors caused concentration-dependent release of [Ca2+]i from internal stores and concentration-dependent increase in glutamate release. Histamine H2 receptor activation increased cyclic adenosine monophosphate (cAMP) levels and phosphorylation of transcription factor cAMP response-element binding protein. Taken together, these data emphasize a role for histamine in neuron-glia communication.

Heparan sulfate in pancreatic ß-cells contributes to normal glucose homeostasis by regulating insulin secretion.

Heparan sulfate (HS), a linear polysaccharide, is involved in diverse biological functions of various tissues. HS is expressed in pancreatic ß-cells and may be involved in ß-cell functions. However, the importance of HS for ß-cell function remains unknown. Here, we generated mice with ß-cell-specific deletion of Ext1 (ßExt1CKO), which encodes an enzyme essential for HS synthesis, to investigate the detailed roles of HS in ß-cell function. ßExt1CKO mice decreased body weights compared with control mice, despite increased food intake. Additionally, ßExt1CKO mice showed impaired glucose tolerance associated with decreased insulin secretion upon glucose challenge. Glucose-induced insulin secretion (GIIS) from isolated ßExt1CKO islets was also significantly reduced, highlighting the contribution of HS to insulin secretion and glucose homeostasis. The gene expression essential for GIIS was decreased in ßExt1CKO islets. Pdx1 and MafA were downregulated in ßExt1CKO islets, indicating that HS promoted ß-cell development and maturation. BrdU- or Ki67-positive ß-cells were reduced in ßExt1CKO pancreatic sections, suggesting the involvement of HS in the proliferation of ß-cells. Moreover, insufficient vascularization in ßExt1CKO islets may contribute to central distribution of α-cells. These data demonstrate HS plays diverse roles in ß-cells, and that loss of HS leads to insufficient insulin secretion and dysregulation of glucose homeostasis.

Histamine N-methyltransferase regulates aggression and the sleep-wake cycle.

Histamine is a neurotransmitter that regulates diverse physiological functions including the sleep-wake cycle. Recent studies have reported that histaminergic dysfunction in the brain is associated with neuropsychiatric disorders. Histamine N-methyltransferase (HNMT) is an enzyme expressed in the central nervous system that specifically metabolises histamine; yet, the exact physiological roles of HNMT are unknown. Accordingly, we phenotyped Hnmt knockout mice (KO) to determine the relevance of HNMT to various brain functions. First, we showed that HNMT deficiency enhanced brain histamine concentrations, confirming a role for HNMT in histamine inactivation. Next, we performed comprehensive behavioural testing and determined that KO mice exhibited high aggressive behaviours in the resident-intruder and aggressive biting behaviour tests. High aggression in KO mice was suppressed by treatment with zolantidine, a histamine H2 receptor (H2R) antagonist, indicating that abnormal H2R activation promoted aggression in KO mice. A sleep analysis revealed that KO mice exhibited prolonged bouts of awakening during the light (inactive) period and compensatory sleep during the dark (active) period. Abnormal sleep behaviour was suppressed by treatment with pyrilamine, a H1R antagonist, prior to light period, suggesting that excessive H1R activation led to the dysregulation of sleep-wake cycles in KO mice. These observations inform the physiological roles of HNMT.

JNJ10181457, a histamine H3 receptor inverse agonist, regulates in vivo microglial functions and improves depression-like behaviours in mice.

Brain histamine acts as a neurotransmitter and regulates various physiological functions, such as learning and memory, sleep-wake cycles, and appetite regulation. We have recently shown that histamine H3 receptor (H3R) is expressed in primary mouse microglia and has a strong influence on critical functions in microglia, including chemotaxis, phagocytosis, and cytokine secretion in vitro. However, the importance of H3R in microglial activity in vivo remains unknown. Here, we examined the effects of JNJ10181457 (JNJ), a selective and potent H3R inverse agonist, on microglial functions ex vivo and in vivo. First, we injected ATP, which is a typical chemoattractant, into hippocampal slices to investigate the effect of JNJ on chemotaxis. ATP-induced microglial migration toward the injected site was significantly suppressed by JNJ treatment. Next, we examined whether JNJ affected microglial phagocytosis in hippocampal slices and in the prefrontal cortex. Microglial engulfment of dead neurons induced by N-methyl-d-aspartate was inhibited in the presence of JNJ. The increase in zymosan particle uptake by activated microglia in the prefrontal cortex was prevented by JNJ administration. Finally, we determined the importance of JNJ in a lipopolysaccharide (LPS)-induced depression model. JNJ reduced the LPS-induced upregulation of microglial pro-inflammatory cytokines and improved depression-like behaviour in the tail-suspension test. These results demonstrate the inhibitory effects of JNJ on chemotaxis, phagocytosis, and cytokine production in microglia inside the brain, and highlight the importance of microglial H3R for brain homeostasis.

Characterization of murine polyspecific monoamine transporters.

The dysregulation of monoamine clearance in the central nervous system occurs in various neuropsychiatric disorders, and the role of polyspecific monoamine transporters in monoamine clearance is increasingly highlighted in recent studies. However, no study to date has properly characterized polyspecific monoamine transporters in the mouse brain. In the present study, we examined the kinetic properties of three mouse polyspecific monoamine transporters [organic cation transporter 2 (Oct2), Oct3, and plasma membrane monoamine transporter (Pmat)] and compared the absolute mRNA expression levels of these transporters in various brain areas. First, we evaluated the affinities of each transporter for noradrenaline, dopamine, serotonin, and histamine, and found that mouse ortholog substrate affinities were similar to those of human orthologs. Next, we performed drug inhibition assays and identified interspecies differences in the pharmacological properties of polyspecific monoamine transporters; in particular, corticosterone and decynium-22, which are widely recognized as typical inhibitors of human OCT3, enhanced the transport activity of mouse Oct3. Finally, we quantified absolute mRNA expression levels of each transporter in various regions of the mouse brain and found that while all three transporters were ubiquitously expressed, Pmat was the most highly expressed transporter. These results provide an important foundation for future translational research investigating the roles of polyspecific monoamine transporters in neurological and neuropsychiatric disease.

Histamine H3 receptor in primary mouse microglia inhibits chemotaxis, phagocytosis, and cytokine secretion.

Histamine is a physiological amine which initiates a multitude of physiological responses by binding to four known G-protein coupled histamine receptor subtypes as follows: histamine H1 receptor (H1 R), H2 R, H3 R, and H4 R. Brain histamine elicits neuronal excitation and regulates a variety of physiological processes such as learning and memory, sleep-awake cycle and appetite regulation. Microglia, the resident macrophages in the brain, express histamine receptors; however, the effects of histamine on critical microglial functions such as chemotaxis, phagocytosis, and cytokine secretion have not been examined in primary cells. We demonstrated that mouse primary microglia express H2 R, H3 R, histidine decarboxylase, a histamine synthase, and histamine N-methyltransferase, a histamine metabolizing enzyme. Both forskolin-induced cAMP accumulation and ATP-induced intracellular Ca(2+) transients were reduced by the H3 R agonist imetit but not the H2 R agonist amthamine. H3 R activation on two ubiquitous second messenger signalling pathways suggests that H3 R can regulate various microglial functions. In fact, histamine and imetit dose-dependently inhibited microglial chemotaxis, phagocytosis, and lipopolysaccharide (LPS)-induced cytokine production. Furthermore, we confirmed that microglia produced histamine in the presence of LPS, suggesting that H3 R activation regulate microglial function by autocrine and/or paracrine signalling. In conclusion, we demonstrate the involvement of histamine in primary microglial functions, providing the novel insight into physiological roles of brain histamine.