Humanin variant P3S is associated with longevity in APOE4 carriers and resists APOE4-induced brain pathology.

The APOE4 allele is recognized as a significant genetic risk factor to Alzheimer's disease (AD) and influences longevity. Nonetheless, some APOE4 carriers exhibit resistance to AD even in advanced age. Humanin, a mitochondrial-derived peptide comprising 24 amino acids, has variants linked to cognitive resilience and longevity. Our research uncovered a unique humanin variant, P3S, specifically enriched in centenarians with the APOE4 allele. Through in silico analyses and subsequent experimental validation, we demonstrated a strong affinity between humanin P3S and APOE4. Utilizing an APOE4-centric mouse model of amyloidosis (APP/PS1/APOE4), we observed that humanin P3S significantly attenuated brain amyloid-beta accumulation compared to the wild-type humanin. Transcriptomic assessments of mice treated with humanin P3S highlighted its potential mechanism involving the enhancement of amyloid beta phagocytosis. Additionally, in vitro studies corroborated humanin P3S's efficacy in promoting amyloid-beta clearance. Notably, in the temporal cortex of APOE4 carriers, humanin expression is correlated with genes associated with phagocytosis. Our findings suggest a role of the rare humanin variant P3S, especially prevalent among individuals of Ashkenazi descent, in mitigating amyloid beta pathology and facilitating phagocytosis in APOE4-linked amyloidosis, underscoring its significance in longevity and cognitive health among APOE4 carriers.

Histamine and Microglia.

Microglia, a category of glial cells in the central nervous system (CNS), have attracted much attention because of their important role in neuroinflammation. Many translational studies are currently ongoing to discover novel drugs targeting microglia for the treatment of various CNS disorders, such as Alzheimer's disease, Parkinson's disease (PD), and depression. Recent studies have shown that brain histamine, a neurotransmitter essential for the regulation of diverse brain functions, controls glial cells and neurons. In vitro studies using primary microglia and microglial cell lines have reported that histamine receptors are expressed in microglia and control microglial functions, including chemotaxis, migration, cytokine secretion, and autophagy. In vivo studies have demonstrated that histamine-related reagents could ameliorate abnormal symptoms in animal models of human diseases, such as amyotrophic lateral sclerosis (ALS), PD, and brain ischemia. Several human studies have revealed alterations in histamine receptor levels in ALS and PD, emphasizing the importance of the CNS histamine system, including histamine-dependent microglial modulation, as a therapeutic target for these disorders. In this review article, we summarize histamine-related research focusing on microglial functions.

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.

The clinical pharmacology of non-sedating antihistamines.

We previously reported on brain H1 receptor occupancy measurements of antihistamines in human brain using [11C]doxepin and positron emission tomography (PET). We proposed the use of brain H1 receptor occupancy to classify antihistamines objectively into three categories of sedating, less-sedating, and non-sedating antihistamines according to their sedative effects. Non-sedating antihistamines are recommended for the treatment of allergies such as pollinosis and atopic dermatitis because of their low penetration into the central nervous system. Physicians and pharmacists are responsible for fully educating patients about the risks of sedating antihistamines from pharmacological points of view. If a sedating antihistamine must be prescribed, its sedative effects should be thoroughly considered before choosing the drug. Non-sedating antihistamines should be preferentially used whenever possible as most antihistamines are equally efficacious, while adverse effects of sedating antihistamines can be serious. This review summarizes the pharmacological properties of clinically useful non-sedating antihistamines from the perspective of histamine function in the CNS.

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.

Role of histamine H3 receptor in glucagon-secreting αTC1.6 cells.

Pancreatic α-cells secrete glucagon to maintain energy homeostasis. Although histamine has an important role in energy homeostasis, the expression and function of histamine receptors in pancreatic α-cells remains unknown. We found that the histamine H3 receptor (H3R) was expressed in mouse pancreatic α-cells and αTC1.6 cells, a mouse pancreatic α-cell line. H3R inhibited glucagon secretion from αTC1.6 cells by inhibiting an increase in intracellular Ca(2+) concentration. We also found that immepip, a selective H3R agonist, decreased serum glucagon concentration in rats. These results suggest that H3R modulates glucagon secretion from pancreatic α-cells.

Insufficient intake of L-histidine reduces brain histamine and causes anxiety-like behaviors in male mice.

L-histidine is one of the essential amino acids for humans, and it plays a critical role as a component of proteins. L-histidine is also important as a precursor of histamine. Brain histamine is synthesized from L-histidine in the presence of histidine decarboxylase, which is expressed in histamine neurons. In the present study, we aimed to elucidate the importance of dietary L-histidine as a precursor of brain histamine and the histaminergic nervous system. C57BL/6J male mice at 8 wk of age were assigned to 2 different diets for at least 2 wk: the control (Con) diet (5.08 g L-histidine/kg diet) or the low L-histidine diet (LHD) (1.28 g L-histidine/kg diet). We measured the histamine concentration in the brain areas of Con diet-fed mice (Con group) and LHD-fed mice (LHD group). The histamine concentration was significantly lower in the LHD group [Con group vs. LHD group: histamine in cortex (means ± SEs): 13.9 ± 1.25 vs. 9.36 ± 0.549 ng/g tissue; P = 0.002]. Our in vivo microdialysis assays revealed that histamine release stimulated by high K(+) from the hypothalamus in the LHD group was 60% of that in the Con group (P = 0.012). However, the concentrations of other monoamines and their metabolites were not changed by the LHD. The open-field tests showed that the LHD group spent a shorter amount of time in the central zone (87.6 ± 14.1 vs. 50.0 ± 6.03 s/10 min; P = 0.019), and the light/dark box tests demonstrated that the LHD group spent a shorter amount of time in the light box (198 ± 8.19 vs. 162 ± 14.1 s/10 min; P = 0.048), suggesting that the LHD induced anxiety-like behaviors. However, locomotor activity, memory functions, and social interaction did not differ between the 2 groups. The results of the present study demonstrated that insufficient intake of histidine reduced the brain histamine content, leading to anxiety-like behaviors in the mice.

Mechanism of the histamine H(3) receptor-mediated increase in exploratory locomotor activity and anxiety-like behaviours in mice.

Histaminergic neurons are activated by histamine H(3) receptor (H(3)R) antagonists, increasing histamine and other neurotransmitters in the brain. The prototype H(3)R antagonist thioperamide increases locomotor activity and anxiety-like behaviours; however, the mechanisms underlying these effects have not been fully elucidated. This study aimed to determine the mechanism underlying H(3)R-mediated behavioural changes using a specific H(3)R antagonist, JNJ-10181457 (JNJ). First, we examined the effect of JNJ injection to mice on the concentrations of brain monoamines and their metabolites. JNJ exclusively increased N(τ)-methylhistamine, the metabolite of brain histamine used as an indicator of histamine release, suggesting that JNJ dominantly stimulates the release of histamine release but not of other monoamines. Next, we examined the mechanism underlying JNJ-induced behavioural changes using open-field tests and elevated zero maze tests. JNJ-induced increase in locomotor activity was inhibited by α-fluoromethyl histidine, an inhibitor of histamine synthesis, supporting that H(3)R exerted its effect through histamine neurotransmission. The JNJ-induced increase in locomotor activity in wild-type mice was preserved in H(1)R gene knockout mice but not in histamine H2 receptor (H(2)R) gene knockout mice. JNJ-induced anxiety-like behaviours were partially reduced by diphenhydramine, an H(1)R antagonist, and dominantly by zolantidine, an H(2)R antagonist. These results suggest that H(3)R blockade induces histamine release, activates H(2)R and elicits exploratory locomotor activity and anxiety-like behaviours.

Predominant role of plasma membrane monoamine transporters in monoamine transport in 1321N1, a human astrocytoma-derived cell line.

Monoamine neurotransmitters should be immediately removed from the synaptic cleft to avoid excessive neuronal activity. Recent studies have shown that astrocytes and neurons are involved in monoamine removal. However, the mechanism of monoamine transport by astrocytes is not entirely clear. We aimed to elucidate the transporters responsible for monoamine transport in 1321N1, a human astrocytoma-derived cell line. First, we confirmed that 1321N1 cells transported dopamine, serotonin, norepinephrine, and histamine in a time- and dose-dependent manner. Kinetics analysis suggested the involvement of low-affinity monoamine transporters, such as organic cation transporter (OCT) 2 and 3 and plasma membrane monoamine transporter (PMAT). Monoamine transport in 1321N1 cells was not Na(+) /Cl(-) dependent but was inhibited by decynium-22, an inhibitor of low-affinity monoamine transporters, which supported the importance of low-affinity transporters. RT-PCR assays revealed that 1321N1 cells expressed OCT3 and PMAT but no other neurotransmitter transporters. Another human astrocytoma-derived cell line, U251MG, and primary human astrocytes also exhibited the same gene expression pattern. Gene-knockdown assays revealed that 1321N1 and primary human astrocytes could transport monoamines predominantly through PMAT and partly through OCT3. These results might indicate that PMAT and OCT3 in human astrocytes are involved in monoamine clearance.

Molecular mechanism of histamine clearance by primary human astrocytes.

Histamine clearance is an essential process for avoiding excessive histaminergic neuronal activity. Previous studies using rodents revealed the predominant role of astrocytes in brain histamine clearance. However, the molecular mechanism of histamine clearance has remained unclear. We detected histamine N-methyltransferase (HNMT), a histamine-metabolizing enzyme, in primary human astrocytes and the astrocytes of human brain specimens. Immunocytochemical analysis and subcellular fractionation assays revealed that active HNMT localized to the cytosol, suggesting that histamine transport into the cytosol is crucial for histamine inactivation. We showed that primary human astrocytes transported histamine in a time-dependent manner. Kinetics analysis showed that two low-affinity transporters were involved in histamine transport. Histamine uptake by primary human astrocytes was not dependent on the extracellular Na(+) /Cl(-) concentration. Histamine is reported to be a substrate for three low-affinity and Na(+) /Cl(-) -independent transporters: organic cation transporter 2 (OCT2), OCT3, and plasma membrane monoamine transporter (PMAT). RT-PCR analysis revealed that OCT3 and PMAT were expressed in primary human astrocytes. Immunohistochemistry confirmed OCT3 and PMAT expression in the astrocytes of human brain specimens. Drug inhibition assays and gene knockdown assays revealed the major contribution of PMAT and the minor contribution of OCT3 to histamine transport. The present study demonstrates for the first time that the molecular mechanism of histamine clearance is by primary human astrocytes. These findings might indicate that PMAT, OCT3 and HNMT in human astrocytes play a role in the regulation of extraneuronal histamine concentration and the activities of histaminergic neurons.