Narcolepsy is strongly associated with the T-cell receptor alpha locus.

Narcolepsy with cataplexy, characterized by sleepiness and rapid onset into REM sleep, affects 1 in 2,000 individuals. Narcolepsy was first shown to be tightly associated with HLA-DR2 (ref. 3) and later sublocalized to DQB1*0602 (ref. 4). Following studies in dogs and mice, a 95% loss of hypocretin-producing cells in postmortem hypothalami from narcoleptic individuals was reported. Using genome-wide association (GWA) in Caucasians with replication in three ethnic groups, we found association between narcolepsy and polymorphisms in the TRA@ (T-cell receptor alpha) locus, with highest significance at rs1154155 (average allelic odds ratio 1.69, genotypic odds ratios 1.94 and 2.55, P < 10(-21), 1,830 cases, 2,164 controls). This is the first documented genetic involvement of the TRA@ locus, encoding the major receptor for HLA-peptide presentation, in any disease. It is still unclear how specific HLA alleles confer susceptibility to over 100 HLA-associated disorders; thus, narcolepsy will provide new insights on how HLA-TCR interactions contribute to organ-specific autoimmune targeting and may serve as a model for over 100 other HLA-associated disorders.

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.

Characterization of peroxidase in buckwheat seed.

A peroxidase (POX)-containing fraction was purified from buckwheat seed. The POX consisted of two isozymes, POX I and POX II, that were purified 6.6- and 67.4-fold, respectively. Their molecular weights were estimated to be 46.1 kDa (POX I) and 58.1 kDa (POX II) by gel filtration. While POX I and II each oxidized quercetin, o-dianisidine, ascorbic acid and guaiacol, only POX II oxidized ABTS. Kinetic studies revealed that POX I and II had lower K(m) values for quercetin (0.071 and 0.028 mM), ABTS (0.016 mM for POX II) and ascorbic acid (0.043 and 0.029 mM) than for o-dianisidine (0.229 and 0.137 mM) and guaiacol (0.288 and 0 ). The optimum pHs of POX I and II for various substrates were almost the same, except for quercetin; pH 8.0 for POX I and pH 4.5 for II. Their optimal temperatures were 30 degrees C (POX I) and 10 degrees C (POX II), and POX I was more stable than POX II above 30 degrees C.

Effects of lipase, lipoxygenase, peroxidase, and rutin on quality deteriorations in buckwheat flour.

To investigate the effects of changes in lipase, lipoxygenase, peroxidase (POX), and rutin concentrations on the quality of buckwheat flour, 14 buckwheat varieties were stored for 0, 4, 10, and 30 days at 5 or 20 degrees C. During the storage period, lipase activity correlated to pH (significantly negative) and water-soluble acid (WSA) (significantly positive). The lipoxygenase 1 protein concentration had a negative correlation to WSA (significant at 0 and 4 storage days at 5 degrees C and at 0 and 10 storage days at 20 degrees C). POX had significant correlation to pH and peroxide value (POV) at 5 degrees C, whereas it was not significant at 20 degrees C. The rutin concentration had negative correlations to WSA (significant at 30 days of storage at 5 degrees C and at 4 days of storage at 20 degrees C). Thus, lipase activity plays an important role that relates to lipid degradation in quality deterioration of buckwheat flour.

Purification and characterization of lipase in buckwheat seed.

To obtain basic information about enzymatic deterioration of buckwheat flour, triacylglycerol lipase (LIP; EC 3.1.1.3) was purified from buckwheat seed. The LIP consisted of two isozymes, LIP I and LIP II, and they were purified with purification folds of 60 and 143 with final specific activities of 0.108 and 0.727 mumol of fatty acid released per minute per milligram of protein at 30 degrees C using triolein as a substrate. Molecular weights were estimated to be 150 (LIP I) and 28.4 kDa (LIP I) by gel filtration and 171 (LIP I) and 26.5 kDa (LIP II) by SDS-PAGE. Optimal pHs of LIP activities were 3.0 (LIP I) and 6.0 (Lip II) using triolein as a substrate. Both LIP I and II reacted in the acidic pH range. Optimal temperatures were 30 (LIP I) and 40 degrees C (LIP II), and both LIP I and II were stable below 30 degrees C when p-nitrophenyl-laurate was used as a substrate. However, they were inactivated above 60 degrees C. On the other hand, when triolein was used as a substrate, optimal temperatures were 30 degrees C for both LIP I and II, and they retained 40% of their activity after a 4 h incubation of enzymes at 70 degrees C. LIP I and II had higher activity against triolein than monoolein or tri/monopalmitin. Most of the LIP activity was distributed in the embryo.