Masashi Yanagisawa

IGFBP3 Colocalizes with and Regulates Hypocretin (Orexin)

Background The sleep disorder narcolepsy is caused by a vast reduction in neurons producing the hypocretin (orexin) neuropeptides. Based on the tight association with HLA, narcolepsy is believed to result from an autoimmune attack, but the cause of hypocretin cell loss is still unknown. We performed gene expression profiling in the hypothalamus to identify novel genes dysregulated in narcolepsy, as these may be the target of autoimmune attack or modulate hypocretin gene expression. Methodology/Principal Findings We used microarrays to compare the transcriptome in the posterior hypothalamus of (1) narcoleptic versus control postmortem human brains and (2) transgenic mice lacking hypocretin neurons versus wild type mice. Hypocretin was the most downregulated gene in human narcolepsy brains. Among many additional candidates, only one, insulin-like growth factor binding protein 3 (IGFBP3), was downregulated in both human and mouse models and co-expressed in hypocretin neurons. Functional analysis indicated decreased hypocretin messenger RNA and peptide content, and increased sleep in transgenic mice overexpressing human IGFBP3, an effect possibly mediated through decreased hypocretin promotor activity in the presence of excessive IGFBP3. Although we found no IGFBP3 autoantibodies nor a genetic association with IGFBP3 polymorphisms in human narcolepsy, we found that an IGFBP3 polymorphism known to increase serum IGFBP3 levels was associated with lower CSF hypocretin-1 in normal individuals. Conclusions/Significance Comparison of the transcriptome in narcolepsy and narcolepsy model mouse brains revealed a novel dysregulated gene which colocalized in hypocretin cells. Functional analysis indicated that the identified IGFBP3 is a new regulator of hypocretin cell physiology that may be involved not only in the pathophysiology of narcolepsy, but also in the regulation of sleep in normal individuals, most notably during adolescence. Further studies are required to address the hypothesis that excessive IGFBP3 expression may initiate hypocretin cell death and cause narcolepsy.

Diabetic endothelin B receptor–deficient rats develop severe hypertension and progressive renal failure

The endothelin (ET) system has been implicated in the pathogenesis of diabetic nephropathy. The role of the ET-B receptor (ETBR) is still unclear. The effect of ETBR deficiency on the progression of diabetic nephropathy in a streptozotocin model was analyzed in four groups: (1) Homozygous ETBR-deficient (ETBRd) diabetic rats, (2) ETBRd rats, (3) diabetic controls, and (4) wild-type controls. BP and kidney function were measured for 10 wk, followed by biochemical and histologic analysis of the kidneys. The study demonstrates that ETBRd diabetic rats on a normal-sodium diet develop severe hypertension, albuminuria, and a mild reduction of creatinine clearance. The strong BP rise seems not to be caused by activation of the renin-angiotensin-aldosterone system or by suppression of the nitric oxide system. Elevated plasma ET-1, possibly reflecting a reduced ETBR-dependent clearance, seems to cause the severe hypertension via the ETA receptor. The results do not support the hypothesis that a reduction of ETBR activity inhibits the progression of diabetic nephropathy. The study demonstrates for the first time that the combination of diabetes and ETBR deficiency causes severe low-renin hypertension with progressive renal failure.

Differential roles of orexin receptor-1 and -2 in the regulation of non-REM and REM sleep

s / Neuroscience Research 71S (2011) e108–e415 e169 measurements using fibroblast cells would be a powerful tool for assessing individual’s circadian properties. Research fund: SRPBS 10036033. doi:10.1016/j.neures.2011.07.728 P2-n01 Ghrelin as a regulator of the circadian body temperature rhythm Kei Nagashima 1,2,3 , Ken Tokizawa 2 1 Integ. Physiol., Fac. Human Sci., Waseda University, Tokorozawa, Japan 2 Acitive Life, Waseda University, Tokorozawa, Japan 3 IABS, Waseda University, Tokorozawa, Japan We have shown that, in mice fasting attenuates thermoregulatory response in a cold environment, and the response differs among time of the day: mice increased metabolic heat production in the dark (active) phase and maintained body temperature (Tb), whereas in the light (inactive) phase, heat production was less, resulting in hypothermia. The aim of the present study was to elucidate whether leptin and ghrelin could modulate those phasedependent differences in thermoregulation. In ob/ob mice, thermoregulatory responses were attenuated in the cold compared with wild-type mice, whereas no differences between the light and dark phases were found. Next, wild-type mice received an i.p. saline or ghrelin injection at Zeitgeber time (ZT) 1 (light phase) or ZT13 (dark phase), and then cold exposure was carried out at ZT2–4 or ZT14–16. During the cold exposure in the light phase, the administration of ghrelin decreased Tb by 1.7 ◦C and induced less increase in oxygen consumption rate than saline-injected mice. In contrast, in the dark phase, Tb in the cold was maintained in ghrelin-injected mice and augmentation of oxygen consumption rate was similar between the treatments. We also found that expression of cFos in the hypothalamus, an indicator of neuronal activation, was significantly enhanced in the suprachiasmatic nucleus after ghrelin administration only in the light phase. During the cold exposure, cFos in the paraventricular nucleus was increased only in the dark phase. These results indicate that ghrelin time-dependently modulates thermoregulation in a cold via hypothalamic mechanisms. Research fund: KAKENHI20390066. doi:10.1016/j.neures.2011.07.729 P2-n02 Carbachol-induced oscillations in rat hippocampal slices are modulated by a circadian rhythm Hiroki Nakatsuka , Kiyohisa Natsume Dept. of Brain Science and Engineering, Grad. Sch. of LSSE, KIT, Kitakyusyu, Japan Brain has circadian rhythm and the rhythm affects the neuronal activities. Cholinergic agent carbachol induces oscillation intermittently in rat hippocampal slices. In the present paper, whether the carbachol-induced oscillation is generated differently in the lightand dark-phase or not was studied. Carbachol induced oscillations in hippocampal slices which are derived from the rat brains in the light and dark phases. The frequency, duration and amplitude of the oscillations in both phase slices are not significantly different. On the other hand, the inter-burst intervals (IBI) in the dark-phase slices were significantly shorter than those in the lightphase slices. The muscarinic receptor antagonist atropine suppressed the carbachol-induced oscillation, while the nicotinic receptor antagonist dtubocrarine significantly decreased the frequency of the oscillation. The GABAA receptor antagonist SR95531 (gabazine) significantly shortened IBI in the light-phase slices, while it did not change it in the dark-phase slices. Melatonin also shortened IBI of the oscillation in the light-phase slices. Because carbachol-induced oscillation generates in CA3 neuronal network, the obtained results suggest that the interneurons in CA3 will activate in the light phase, and modulate the neuronal rhythm in hippocampus depending on the light and dark cycle. Research fund: Challenging Exploratory Research (20650094). doi:10.1016/j.neures.2011.07.730 P2-n03 c-Jun N-terminal kinase 3 regulates circadian period and photic response of behavioral rhythms in mice Sato Honma 1 , Shin-ya Nishide 1, Daisuke Ono 1, Hiroto Nakajima 2, Naoya Shinozaki 2, Hirokazu Matsuki 2, Naoya Wada 2, Hirofumi Doi 3, Ken-ichi Honma 1 1 Dep. Physiology, Grad. Sch. of Med. Hokkaido Univ., Sapporo, Japan 2 R&D Division, Daiichi Sankyo Co. Ltd., Japan 3 Celester Lexico-Sciences, Inc., Japan c-Jun N-terminal kinase (JNK), a member of MAP kinase family, is expressed in the hypothalamic suprachiasmatic nucleus (SCN), the site of mammalian master circadian clock, and activated in a circadian fashion and in response to photic stimuli. Among three isoforms of JNK, JNK3 is expressed almost exclusively in the central nervous system and has been implicated in several neurodegenerative diseases. In order to examine the role of JNK3 in circadian time keeping system, we measured behavioral rhythms of JNK3 deficient mice in response to various lighting conditions. Spontaneous locomotor activity of adult male JNK3 deficient mice of C57BL/6J background (KO, n = 22) and wild type mice (WT, n = 16) was measured by an infrared thermal sensor, first in a light–dark cycle (LD), then in continuous darkness (DD) for more than 100 days. A light pulse of 30 min, 300 lux, was given at the different phases of free running behavioral rhythms in DD, and phase-dependency of phase-shifts and period-changes were examined. All KO showed robust circadian behavioral rhythms, but their free running period in DD was significantly longer in KO than in WT. Circadian rhythms in clock gene expression were altered in the SCN of KO. Phase-shifts of behavioral rhythms by light pulse were significantly reduced in KO. Especially, phase-advance shifts were markedly reduced in KO by a light pulse during the subjective morning with a lack of concomitant period changes. The results suggest that JNK3 regulates the oscillation speed and photic responses of the circadian clock. Reduced frequency modulation in KO suggests the adaptive role of JNK3 in 24 h light–dark cycle. Research fund: KAKENH (I21390064). doi:10.1016/j.neures.2011.07.731 P2-n04 Differential roles of orexin receptor-1 and -2 in the regulation of non-REM and REM sleep Emi Hasegawa 1 , Michihiro Mieda 1, Yaz Kisanuki 2, Masashi Yanagisawa 2, Takeshi Sakurai 1 1 Grad. Sch. Med. Kanazawa Univ., Kanazawa, Japan 2 Southwestern Med. Center Univ. Texas, Dallas, USA Orexin-A and orexin-B are hypothalamic neuropeptides that play critical roles in the maintenance of wakefulness. Intracerebroventricular (ICV) administration of orexin-A has been shown to promote wakefulness and suppress both rapid eye movement (REM) sleep and non-REM (NREM) sleep through the orexin receptor-1 (OX1R) and orexin receptor-2 (OX2R). Here, we elucidated the differential roles of orexin receptors in the regulation of sleep and wakefulness by comparing the effects of ICV orexin-A administration in wild-type, OX1R−/− and OX2R−/− mice. The effects of orexin-A on wakefulness and NREM sleep were significantly attenuated in both knockout mice as compared to wild-type mice, with substantially larger attenuation in OX2R−/− mice than in OX1R−/− mice. These results suggest that although the OX2R-mediated pathway has a pivotal role in the promotion of wakefulness, OX1R also plays additional roles in promoting arousal. In contrast, suppression of REM sleep by orexin-A administration was slightly and similarly attenuated in both OX1R−/− and OX2R−/− mice, suggesting a comparable contribution of the two receptors to REM sleep suppression. Histological studies demonstrated differential distributions of each receptor subtype in distinct neuronal populations with specific neurotransmitter identities in brainstem cholinergic/monoaminergic neurons. In the laterodorsal tegmental and pedunculopontine tegmental nuclei especially, cholinergic neurons exclusively expressed OX1R mRNA, but OX2R mRNA was expressed mainly in GABAergic putative interneurons. Thus, each orexin receptor subtype plays differential roles in gating NREM and REM sleep, through distinct neuronal pathways. doi:10.1016/j.neures.2011.07.732

Urinary protein profiling with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry in ETB receptor-deficient rats

The pathways leading to salt-sensitive hypertension and renal damage in rescued ETB receptor-deficient (ETBRd) rats are still unknown. The objective of the study was therefore to identify modifications of urinary peptide and protein expression in ETBRd rats (n = 9) and wild-type controls (n = 6) using SDS - polyacrylamide gel electrophoresis (SDS-PAGE) and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. Glomerular filtration rate, glomerulosclerosis, and tubulointerstitial fibrosis did not differ between the groups. ETBRd rats showed slightly higher blood pressure (p < 0.001), media/lumen ratio of intrarenal arteries (p < 0.01), and albuminuria (p < 0.01). SDS-PAGE confirmed albuminuria, but showed no differences in the urinary excretion of low molecular weight proteins (<60 kDa). SELDI-TOF-MS profiling revealed 9 proteomic features at molecular masses (Da) of 2720, 2980, 3130, 3345, 6466, 6682, 8550, 18 729, and 37 492, which were significantly elevated (p < 0.02) in urine of ETBRd rats. The results demonstrate that, independent of structural changes in the kidneys, ETB-receptor deficiency causes specific differences in urinary peptide and protein excretion. SELDI-TOF-MS may be a valuable tool for the characterization of urinary biomarkers helping to uncover the mechanism of ETBR action in the kidney.