Involvement of PLAGL1/ZAC1 in hypocretin/orexin transcription.

The hypocretin/orexin neuropeptide system coordinates the regulation of various physiological processes. Our previous study reported that a reduction in the expression of pleomorphic adenoma gene­like 1 (Plagl1), which encodes a C2H2 zinc­finger transcription factor, occurs in hypocretin neuron­ablated transgenic mice, suggesting that PLAGL1 is co­expressed in hypocretin neurons and regulates hypocretin transcription. The present study examined whether canonical prepro­hypocretin transcription is functionally modulated by PLAGL1. Double immunostaining indicated that the majority of hypocretin neurons were positive for PLAGL1 immunoreactivity in the nucleus. Notably, PLAGL1 immunoreactivity in hypocretin neurons was altered in response to several conditions affecting hypocretin function. An uneven localization of PLAGL1 was detected in the nuclei of hypocretin neurons following sleep deprivation. Chromatin immunoprecipitation revealed that endogenous PLAGL1 may bind to a putative PLAGL1­binding site in the proximal region of the hypocretin gene, in the murine hypothalamus. In addition, electroporation of the PLAGL1 expression vector into the fetal hypothalamus promoted hypothalamic hypocretin transcription. These results suggested that PLAGL1 may regulate hypothalamic hypocretin transcription.

Anti-Tribbles Pseudokinase 2 (TRIB2)-Immunization Modulates Hypocretin/Orexin Neuronal Functions.

Study Objectives: Recent findings showed that 16%-26% of narcolepsy patients were positive for anti-tribbles pseudokinase 2 (TRIB2) antibody, and the intracerebroventricular administration of immunoglobulin-G purified from anti-TRIB2 positive narcolepsy patients caused hypocretin/orexin neuron loss. We investigated the pathophysiological role of TRIB2 antibody using TRIB2-immunized rats and hypocretin/ataxin-3 transgenic (ataxin-3) mice. Methods: Plasma, cerebrospinal fluid (CSF), and hypothalamic tissues from TRIB2-immunized rats were collected. Anti-TRIB2 titers, hypocretin contents, mRNA expressions, the cell count of hypocretin neurons, and immunoreactivity of anti-TRIB2 antibodies on hypocretin neurons were investigated. The plasma from ataxin-3 mice was also used to determine the anti-TRIB2 antibody titer changes following the loss of hypocretin neurons. Results: TRIB2 antibody titers increased in the plasma and CSF of TRIB2-immunized rats. The hypothalamic tissue immunostained with the sera from TRIB2-immunized rats revealed positive signals in the cytoplasm of hypcretin neurons. While no changes were found regarding hypothalamic hypocretin contents or cell counts, but there were significant decreases of the hypocretin mRNA level and release into the CSF. The plasma from over 26-week-old ataxin-3 mice, at the advanced stage of hypocretin cell destruction, showed positive reactions against TRIB2 antigen, and positive plasma also reacted with murine hypothalamic hypocretin neurons. Conclusions: Our results suggest that the general activation of the immune system modulates the functions of hypocretin neurons. The absence of a change in hypocretin cell populations suggested that factors other than anti-TRIB2 antibody play a part in the loss of hypocretin neurons in narcolepsy. The increased anti-TRIB2 antibody after the destruction of hypocretin neurons suggest that anti-TRIB2 antibody in narcolepsy patients is the consequence rather than the inciting cause of hypocretin cell destruction.

Hypothalamo­hypophysial system in rats with autotransplantation of the adrenal cortex.

Patients with bilateral pheochromocytoma often require an adrenalectomy. Autotransplantation of the adrenal cortex is an alternative therapy that could potentially be performed instead of receiving glucocorticoid replacement following adrenalectomy. Adrenal cortex autotransplantation aims to avoid the side effects of long­term steroid treatment and adrenal insufficiency. Although the function of the hypothalamo­hypophysial system is critical for patients who have undergone adrenal cortex autotransplantation, the details of that system, with the exception of adrenocorticotropic hormone in the subjects with adrenal autotransplantation, have been overlooked for a long time. To clarify the precise effect of adrenal autotransplantation on the pituitary gland and hypothalamus, the current study examined the gene expression of hormones produced from the hypothalamus and pituitary gland. Bilateral adrenalectomy and adrenal autotransplantation were performed in 8 to 9­week­old male rats. The hypothalamus and pituitary tissues were collected at 4 weeks after surgery. Transcriptional regulation of hypothalamic and pituitary hormones was subsequently examined by reverse transcription­quantitative polymerase chain reaction. Proopiomelanocortin, glycoprotein hormone α polypeptide, and thyroid stimulating hormone ß were significantly elevated in the pituitary gland of autotransplanted rats when compared with sham­operated rats. In addition, there were significant differences in the levels of corticotropin releasing hormone receptor 1 (Crhr1), Crhr2, nuclear receptor subfamily 3 group C member 1 and thyrotropin releasing hormone receptor between the sham­operated rats and autotransplanted rats in the pituitary gland. In the hypothalamus, corticotropin releasing hormone and urocortin 2 mRNA was significantly upregulated in autotransplanted rats compared with sham­operated rats. The authors identified significant alterations in the function of not only the hypothalamus­pituitary­adrenal axis, but also the adenohypophysis thyrotropes in autotransplanted rats. In the future, it will be important to examine other tissues affected by glucocorticoids following adrenal cortex autotransplantation.

Hypocretin/orexin loss changes the hypothalamic immune response.

Hypocretin, also known as orexin, maintains the vigilance state and regulates various physiological processes, such as arousal, sleep, food intake, energy expenditure, and reward. Previously, we found that when wild-type mice and hypocretin/ataxin-3 littermates (which are depleted of hypothalamic hypocretin-expressing neurons postnatally) were administered lipopolysaccharide (LPS), the two genotypes exhibited significant differences in their sleep/wake cycle, including differences in the degree of increase in sleep periods and in recovery from sickness behaviour. In the present study, we examined changes in the hypothalamic vigilance system and in the hypothalamic expression of inflammatory factors in response to LPS in hypocretin/ataxin-3 mice. Peripheral immune challenge with LPS affected the hypothalamic immune response and vigilance states. This response was altered by the loss of hypocretin. Hypocretin expression was inhibited after LPS injection in both hypocretin/ataxin-3 mice and their wild-type littermates, but expression was completely abolished only in hypocretin/ataxin-3 mice. Increases in the number of histidine decarboxylase (HDC)-positive cells and in Hdc mRNA expression were found in hypocretin/ataxin-3 mice, and this increase was suppressed by LPS. Hypocretin loss did not impact the change in expression of hypothalamic inflammatory factors in response to LPS, except for interferon gamma and colony stimulating factor 3. The number of c-Fos-positive/HDC-positive cells in hypocretin/ataxin-3 mice administered LPS injections was elevated, even during the rest period, in all areas, suggesting that there is an increase in the activity of histaminergic neurons in hypocretin/ataxin-3 mice following LPS injection. Taken together, our results suggest a novel role for hypocretin in the hypothalamic response to peripheral immune challenge. Our findings contribute to the understanding of the pathophysiology of narcolepsy.

Passive transfer of narcolepsy: anti-TRIB2 autoantibody positive patient IgG causes hypothalamic orexin neuron loss and sleep attacks in mice.

Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness and cataplexy (a sudden weakening of posture muscle tone usually triggered by emotion) caused by the loss of orexin neurons in the hypothalamus. Autoimmune mechanisms are implicated in narcolepsy by increased frequency of specific HLA alleles and the presence of specific autoantibody (anti-Tribbles homolog 2 (TRIB2) antibodies) in the sera of patients with narcolepsy. Presently, we passively transferred narcolepsy to naïve mice by injecting intra-cerebra-ventricularly (ICV) pooled IgG positive for anti-TRIB2 antibodies. Narcolepsy-IgG-injected mice had a loss of the NeuN (neuronal marker), synaptophysin (synaptic marker) and orexin-positive neurons in the lateral hypothalamus area in narcolepsy compared to control-IgG-injected mice and these changes were associated with narcolepsy-like immobility attacks at four weeks post injection and with hyperactivity and long term memory deficits in the staircase and novel object recognition tests. Similar behavioral and cognitive deficits are observed in narcoleptic patients. This is the first report of passive transfer of experimental narcolepsy to naïve mice induced by autoantibodies and supports the autoimmune pathogenesis in narcolepsy.

Identification of alkylbenzene sulfonate surfactants leaching from an acrylonitrile butadiene rubber as novel inhibitors of calcineurin activity.

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM) dependent serine/threonine protein phosphatase and plays important role in several cellular functions in both higher and lower eukaryotes. Here we report inhibition of CN by linear alkylbenzene sulfonate. The clue to the finding was obtained while identifying the inhibitory material leaching from acrylonitrile butadiene rubber used for packing. Using standard dodecylbenzene sulfonate (C12-LAS), we obtained strong inhibition of CN with a half maximal inhibitory concentration of 9.3 µM, whereas analogs such as p-octylbenzene sulfonate and SDS hardly or only slightly affected CN activity. Three alkaline phosphatases, derived from shrimp, bacteria, and calf-intestine, which exhibit similar enzymatic activities to CN, were not inhibited by C12-LAS at concentrations of up to 100 µM. Furthermore, C12-LAS did not inhibit Ca(2+)/CaM-dependent myosin light chain kinase activity when tested at concentrations of up to 36 µM. The results indicate that C12-LAS is a potent selective inhibitor of CN activity.

Transcriptional regulation of the hypocretin/orexin gene by NR6A1.

The hypocretin (also known as orexin) neuropeptide system coordinates the regulation of various physiological processes. A reduction in Nr6a1 expression was observed in hypocretin neuron-ablated transgenic mice. To show that prepro-hypocretin transcription is functionally modulated by NR6A1, we performed chromatin immunoprecipitation (ChIP) analysis, double-immunostaining, a luciferase reporter assay, and an in utero electroporation study. ChIP analysis showed that endogenous NR6A1 binds to a putative NR6A1-binding site. Double-immunostaining indicated almost all hypocretin neurons were positive for NR6A1 immunoreactivity. NR6A1 overexpression in SH-SY5Y cells modulated hypocretin promoter activity, an effect that was countered by lacking a putative NR6A1-binding site. Electroporation with Nr6a1 in the foetal hypothalamus promoted hypocretin transcription as compared to GFP-electroporation. These experiments confirmed that NR6A1 works as a regulator for hypocretin transcription.

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.

Detection of autoantibodies against hypocretin, hcrtrl, and hcrtr2 in narcolepsy: anti-Hcrt system antibody in narcolepsy.

STUDY OBJECTIVES: The impairment of hypocretin neurotransmission system is considered to play a major role in the pathophysiology of narcolepsy. It has been hypothesized that autoimmune abnormalities underlie the etiology of narcolepsy, based on the tight association with HLA-DRB1*1501/ DQB1*0602. It remains unclear if autoantibodies against hypocretin receptors (hcrtrl and hcrtr2) are involved in narcolepsy. DESIGN: We have developed a novel radioligand binding assay to address this question. Sera from 181 patients with narcolepsy, 10 patients with other hypersomnias, and 91 control subjects were used. Human [35S]-Hcrt, hcrtrl, and hcrtr2 were synthesized by in vitro transcription/translation system. The immune complex of autoantibody and each [35S]-protein were immunoprecipitated and quantified using a radioligand-binding assay. RESULTS: We detected autoantibodies against hypocretin in 3 patients, hcrtrl in 1 patient, and hcrtr2 in 5 patients with narcolepsy. Positive reactions were also found against hcrtrl in 2 and hcrtr2 in 1 control subjects. No relationships were found between these autoantibodies and HLA-DRB1*1501/DQB1*0602 haplotypes, presence of cataplexy, presence of subjective nocturnal sleep disruption, or the score on the Epworth Sleepiness Scale. CONCLUSIONS: Although we have detected autoantibodies against the hypocretin neurotransmission system, our results do not support the hypothesis that autoantibody-mediated dysfunction in the hypocretin system underlies the pathophysiology of narcolepsy.

Zinc ions suppress mitogen-activated interleukin-2 production in Jurkat cells.

Calcineurin (CN) is thought to play an important role in the immune system by regulating cytokine production, for example, interleukin-2 (IL-2) in T-lymphocytes. We have previously shown that physiological concentrations of Zn2+ inhibit CN activity in vitro [K. Takahashi, E. Akaishi, Y. Abe, R. Ishikawa, S. Tanaka, K. Hosaka, Y. Kubohara, Zinc inhibits calcineurin activity in vitro by competing with nickel, Biochem. Biophys. Res. Commun. 307 (2003) 64-68], in spite of the fact that Zn2+ is an essential element of the CN catalytic domain. In this study, in order to assess whether Zn2+ regulates (suppresses) CN activity in vivo and whether Zn2+ can be used as an anti-inflammatory and/or immunosuppressive drug, we examined the effects of Zn2+ on IL-2 production induced by the mitogen, concanavalin A (ConA), in Jurkat T-cells. Zn2+ at 0.2 mM suppressed ConA-induced IL-2 accumulation in the medium of an in vitro culture of Jurkat cells. Zn2+ at 0.03-0.3 mM dose-dependently suppressed ConA-induced IL-2 mRNA expression in Jurkat cells. Zn2+ also suppressed IL-2 mRNA expression induced by phorbol ester (PMA) and ionomycin. Furthermore, Zn2+ and the immunosuppressant FK506 showed an additive inhibitory effect on ConA-induced IL-2 mRNA expression. These results suggest that exogenously added Zn2+ may disturb (increase) the intracellular Zn2+ concentration and inhibit CN activity, thereby suppressing IL-2 production in Jurkat cells. The present study further indicates that Zn2+ may have therapeutic potential in the treatment of T-cell related inflammation and also that Zn2+ may be utilized as a supplemental drug with FK506.