Mie Sakata

Minireview: Sleep regulation in adenosine A2A receptor-deficient mice

Adenosine is proposed to be an endogenous sleep-promoting substance based on the results of a variety of pharmacologic and behavioral experiments.1 For example, sleep is induced in rats after administration of metabolically stable adenosine analogues, such as N6-l-(phenylisopropyl)-adenosine, adenosine-5′-N-ethylcarboxamide, and cyclohexyladenosine,2,3⇓ which are agonists for adenosine A1 receptor (A1R) or A2A receptors (A2ARs). Caffeine is considered to inhibit sleep by acting as an antagonist of adenosine receptor.4 Adenosine content is increased in the basal forebrain, one of the sleep centers, after sleep deprivation and is proposed to be a sleep substance accumulating in the brain during prolonged wakefulness.5 Most previous studies on sleep regulation by adenosine have focused on the A1R-mediated pathway1,6⇓ because A1R is widely distributed in the CNS, whereas A2AR is localized mainly in the striatum, nucleus accumbens, and olfactory bulb. However, we found that A2AR is also important in sleep regulation by using several A1R and A2AR agonists, including N6-cyclopentyladenosine (CPA) and 2-(4-(2-carboxyethyl)phenylethylamino)-adenosine-5′- N -ethylcarboxamideadenosine (CGS 21680).7-12⇓⇓⇓⇓⇓ CGS 21680 is highly selective for the A2AR (Ki = 14 nmol/L), having a much lower affinity for the A1R (Ki = 2,600 nmol/L), whereas CPA is selective for the A1R (Ki = 0.6 nmol/L) and has a lower affinity for the A2AR (Ki = 462 nmol/L).13,14⇓ We investigated the molecular mechanism of induction of non-REM (NREM) sleep by prostaglandin (PG) D2, which is also known as a potent endogenous sleep-promoting substance.15-17⇓⇓ In the course of this study, Satoh et al.7 found that PGD2 …

Aire-expressing thymic medullary epithelial cells originate from β5t-expressing progenitor cells

The thymus provides multiple microenvironments that are essential for the development and repertoire selection of T lymphocytes. The thymic cortex induces the generation and positive selection of T lymphocytes, whereas the thymic medulla establishes self-tolerance among the positively selected T lymphocytes. Cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) constitute the major stromal cells that structurally form and functionally characterize the cortex and the medulla, respectively. cTECs and mTECs are both derived from the endodermal epithelium of the third pharyngeal pouch. However, the molecular and cellular characteristics of the progenitor cells for the distinct TEC lineages are unclear. Here we report the preparation and characterization of mice that express the recombinase Cre instead of β5t, a proteasome subunit that is abundant in cTECs and not detected in other cell types, including mTECs. By crossing β5t-Cre knock-in mice with loxP-dependent GFP reporter mice, we found that β5t-Cre–mediated recombination occurs specifically in TECs but not in any other cell types in the mouse. Surprisingly, in addition to cTECs, β5t-Cre-loxP–mediated GFP expression was detected in almost all mTECs. These results indicate that the majority of mTECs, including autoimmune regulator-expressing mTECs, are derived from β5t-expressing progenitor cells.

Lymphotoxin β Receptor Regulates the Development of CCL21-Expressing Subset of Postnatal Medullary Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play a pivotal role in the establishment of self-tolerance in T cells by ectopically expressing various tissue-restricted self-Ags and by chemoattracting developing thymocytes. The nuclear protein Aire expressed by mTECs contributes to the promiscuous expression of self-Ags, whereas CCR7-ligand (CCR7L) chemokines expressed by mTECs are responsible for the attraction of positively selected thymocytes. It is known that lymphotoxin signals from the positively selected thymocytes preferentially promote the expression of CCR7L rather than Aire in postnatal mTECs. However, it is unknown how lymphotoxin signals differentially regulate the expression of CCR7L and Aire in mTECs and whether CCR7L-expressing mTECs and Aire-expressing mTECs are distinct populations. In this study, we show that the majority of postnatal mTECs that express CCL21, a CCR7L chemokine, represent an mTEC subpopulation distinct from the Aire-expressing mTEC subpopulation. Interestingly, the development of CCL21-expressing mTECs, but not Aire-expressing mTECs, is impaired in mice deficient in the lymphotoxin β receptor. These results indicate that postnatal mTECs consist of heterogeneous subsets that differ in the expression of CCL21 and Aire, and that lymphotoxin β receptor regulates the development of the CCL21-expressing subset rather than the Aire-expressing subset of postnatal mTECs.

Mesolimbic dopaminergic system is involved in diurnal blood pressure regulation.

Parkinsons disease (PD) patients with autonomic failure show no nocturnal decrease in blood pressure (BP). At present, it is not clear if this symptom is attributable to the disturbance of the dopaminergic (DA) system that is responsible for PD. In the present study, we determined that the mesolimbic DA system is involved in diurnal profiles of the mean BP (MBP) by destroying the A10 DA system in rats with 6-hydroxydopamine. In control rats, a clear dip in the MBP and heart rate (HR) occurs during the light, that is, resting period, analogous to the nocturnal dip in normal humans. This normal daytime decrease in MBP and HR was disturbed by inducing a lesion of the ventral tegmental area (VTA) DA neurons, although the rhythms of wake-sleep duration and behavioral activity remained relatively intact. On the basis of this evidence, the absence of a nocturnal dip in BP in PD patients is attributed to impairment of the mesolimbic DA system.

Thymic nurse cells provide microenvironment for secondary T cell receptor α rearrangement in cortical thymocytes

Distinct subsets of thymic epithelial cells (TECs) support T-cell development and selection. Isolated TECs contain multicellular complexes that enclose many viable thymocytes. However, the functions of those TECs, termed thymic nurse cells (TNCs), are unclear and the idea that TNCs are present in vivo is questioned. Here, we show that TNCs represent a fraction of cortical (c)TECs that are defined by the expression of thymoproteasomes. Intravital imaging revealed TNCs in the thymic cortex in situ, whereas TNCs were detected neither during embryogenesis nor in the postnatal thymuses of various “positive-selector” T-cell receptor (TCR)-transgenic mice, indicating that TNCs are not essential for T-cell differentiation, including positive selection. Rather, cells within TNCs were enriched for long-lived CD4+CD8+ thymocytes that underwent secondary TCR-Vα rearrangement. Thus, TNC complexes are formed in vivo by persistent cTEC–thymocyte interactions that then provide a microenvironment that optimizes T-cell selection through secondary TCR rearrangement.

Role of macrophage-derived hypoxia-inducible factor (HIF)-1α as a mediator of vascular remodelling.

AIMS Excessive vascular remodelling leads to progression of a wide range of vasculopathies, and the immune response to intimal injuries is crucial in this process. This vascular remodelling occurs in the hypoxic microenvironment and is closely related to the immune system. Macrophages play a key role in immunological-cell-mediated arterial remodelling. In this study, we clarified the role of macrophage-derived hypoxia-inducible factor (HIF-1α) in vascular remodelling. METHODS AND RESULTS Wire-induced femoral arterial injury was inflicted in mice lacking the macrophage-specific HIF-1α gene and in their wild-type counterparts. The mutant mice showed both suppressed wire-induced neointimal thickening and decreased infiltration of inflammatory cells in the adventitia, compared with wild-type mice. Studies to clarify the mechanism of restrained vascular remodelling in the mutant mice revealed decreased production of pro-inflammatory cytokines by the activated macrophages and suppressed macrophage migration activity in the mutant mice. Gene expressions of the HIF-1α-deficient macrophages positively correlated with the phenotypic profile of M2 macrophages and negatively correlated with that of M1 macrophages. CONCLUSION Our results show that HIF-1α in macrophages plays a crucial role in promoting vascular inflammation and remodelling. As decreasing HIF-1α activity in macrophages may prevent the progression of vascular remodelling, HIF-1α may be a possible therapeutic target in vascular diseases.

Semi-quantitative RT-PCR analysis of environmental influence on P450scc and PNMT mRNA expression in rat adrenal glands

Environmental influence on brain function, particularly spatial learning and memory, has been extensively investigated, but little is known about the influence of environmental conditions on the functions of peripheral organs. In the present study, the effects of different housing conditions on the steady-state levels of mRNAs encoding cholesterol side-chain cleavage enzyme (cytochrome P450scc) and phenylethanolamine N-methyltransferase (PNMT) in adrenal glands was examined to investigate the environmental influence on both adrenocortical and adrenomedullary functions. Behavioral changes of the animals housed in different conditions were first examined to assess the relevance of environmental manipulation used. In consistent with previous findings, housing of the animals in enriched conditions resulted in the significant reduction of spontaneous motor activity (locomotor activity and rearing) in comparison with housing in isolated conditions, thus indicating the relevance of housing conditions used in this work for investigating the environmental influence on adrenal function. Then, the effects of these housing conditions on P450scc and PNMT mRNA levels in adrenal glands were examined using semi-quantitative RT-PCR method. In comparison with the isolated group, the enriched group showed significantly higher levels of P450scc mRNA. In contrast, PNMT mRNA levels in the enriched group were significantly lower than those in the isolated group. These results propose the possibility that the environmental conditions may cause differential alterations in adrenocortical and adrenomedullary functions, although their possible association with behavioral changes still remains to be elucidated.

Arterial pressure and heart rate increase during REM sleep in adenosine A2A-receptor knockout mice, but not in wild-type mice.

Rapid eye movement (REM)-sleep related changes in arterial pressure (AP) and heart rate (HR) were observed in homozygous and heterozygous adenosine A2A receptor (A2AR) knockout (KO) mice, and the corresponding wild-type mice. During REM sleep, the mean AP (MAP) and HR were clearly increased in the homozygous A2AR KO mice, while, in the wild-type mice, they were decreased or maintained at the same level. Neither homozygous nor heterozygous A2AR KO mice showed significant difference in diurnal pattern and the hourly values of MAP and HR compared to the wild-type mice. From these findings, it is likely that the adenosine A2AR is involved in autonomic regulation during REM sleep.

Increased number of Hassall's corpuscles in myasthenia gravis patients with thymic hyperplasia

The thymus is implicated as an organ that contributes to autoimmunity in myasthenia gravis (MG) patients. Hassalls corpuscles (HCs) are assumed to represent the terminally differentiated stage of medullary thymic epithelial cells (mTECs). By using multicolor immunohistofluorescence analysis, we examined HCs in thymuses that were therapeutically excised from MG (+) and MG (-) patients. We found that the number of HCs per unit area of the thymic medulla was significantly elevated in the thymuses of MG (+) patients with thymic hyperplasia. CCL21 expression increased in the hyperplastic MG thymuses. We speculate that the altered differentiation of mTECs is associated with the thymic hyperplasia and the onset of MG.

Foxn1-β5t transcriptional axis controls CD8+ T-cell production in the thymus.

The thymus is an organ that produces functionally competent T cells that protect us from pathogens and malignancies. Foxn1 is a transcription factor that is essential for thymus organogenesis; however, the direct target for Foxn1 to actuate thymic T-cell production is unknown. Here we show that a Foxn1-binding cis-regulatory element promotes the transcription of β5t, which has an essential role in cortical thymic epithelial cells to induce positive selection of functionally competent CD8+ T cells. A point mutation in this genome element results in a defect in β5t expression and CD8+ T-cell production in mice. The results reveal a Foxn1-β5t transcriptional axis that governs CD8+ T-cell production in the thymus.