High level of orexin A observed in the phenylketonuria mouse brain is due to the abnormal expression of prepro-orexin

Orexins/hypocretins are recently discovered neuropeptides, synthesized mainly in the lateral hypothalamus of the brain. Orexins regulate various functions including sleep and apetite. We recently reported increased amount of orexin A in the phenylketonuria (PKU) mouse brain. Whether this is caused by overexpression of the precursor for orexins, prepro-orexin was studied in the PKU mouse brain. Microarray expression analysis revealed overexpression of orexin gene in the brain of PKU mouse. Quantitative real-time RT-PCR showed increased level of prepro-orexin mRNA in the PKU mouse brain. In addition, expression of genes associated with cell signal and growth regulation was also affected in the PKU mouse brain, as observed by microarray analysis. These data suggest that up-regulation of orexin mRNA expression is the possible factor for inducing high orexin A in the brain of PKU mouse. The metabolic environment in the brain of PKU mouse affects normal expression of other genes possibly to result in pathophysiology seen in the PKU mouse, if documented also in patients with PKU.     

Expression of calpastatin, minopontin, NIPSNAP1, rabaptin-5 and neuronatin in the phenylketonuria (PKU) mouse brain: possible role on cognitive defect seen in PKU

Phenylketonuria (PKU) is an inborn error of amino acid metabolism. Phenylalanine hydroxylase (PAH) deficiency results in accumulation of phenylalanine (Phe) in the brain and leads to pathophysiological abnormalities including cognitive defect, if Phe diet is not restricted. Neuronatin and 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) reportedly have role in memory. Therefore, gene expression was examined in the brain of mouse model for PKU. Microarray expression analysis revealed reduced expression of calpastatin, NIPSNAP 1, rabaptin-5 and minopontin genes and overexpression of neuronatin gene in the PKU mouse brain. Altered expression of these genes was further confirmed by one-step real time RT-PCR analysis. Western blot analysis of the mouse brain showed reduced levels of calpastatin and rabaptin-5 and higher amount of neuronatin in PKU compared to the wild type. These observations in the PKU mouse brain suggest that altered expression of these genes resulting in abnormal proteome. These changes in the PKU mouse brain are likely to contribute cognitive impairment seen in the PKU mouse, if documented also in patients with PKU.     

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH^enu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH^enu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH^enu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH^enu2 mice were not protective against cancer.     

Large Neutral Amino Acid Supplementation Exerts Its Effect through Three Synergistic Mechanisms: Proof of Principle in Phenylketonuria Mice

Background

Phenylketonuria (PKU) was the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. However, despite this effect, neuropsychological outcome in PKU still remains suboptimal and the phenylalanine-restricted diet is very demanding. To improve neuropsychological outcome and relieve the dietary restrictions for PKU patients, supplementation of large neutral amino acids (LNAA) is suggested as alternative treatment strategy that might correct all brain biochemical disturbances caused by high blood phenylalanine, and thereby improve neurocognitive functioning.

Objective

As a proof-of-principle, this study aimed to investigate all hypothesized biochemical treatment objectives of LNAA supplementation (normalizing brain phenylalanine, non-phenylalanine LNAA, and monoaminergic neurotransmitter concentrations) in PKU mice.

Methods

C57Bl/6 Pah-enu2 (PKU) mice and wild-type mice received a LNAA supplemented diet, an isonitrogenic/isocaloric high-protein control diet, or normal chow. After six weeks of dietary treatment, blood and brain amino acid and monoaminergic neurotransmitter concentrations were assessed.

Results

In PKU mice, the investigated LNAA supplementation regimen significantly reduced blood and brain phenylalanine concentrations by 33% and 26%, respectively, compared to normal chow (p<0.01), while alleviating brain deficiencies of some but not all supplemented LNAA. Moreover, LNAA supplementation in PKU mice significantly increased brain serotonin and norepinephrine concentrations from 35% to 71% and from 57% to 86% of wild-type concentrations (p<0.01), respectively, but not brain dopamine concentrations (p = 0.307).

Conclusions

This study shows that LNAA supplementation without dietary phenylalanine restriction in PKU mice improves brain biochemistry through all three hypothesized biochemical mechanisms. Thereby, these data provide proof-of-concept for LNAA supplementation as a valuable alternative dietary treatment strategy in PKU. Based on these results, LNAA treatment should be further optimized for clinical application with regard to the composition and dose of the LNAA supplement, taking into account all three working mechanisms of LNAA treatment.     

Is It Possible to Improve Memory Function by Upregulation of the Cholesterol 24S-Hydroxylase (CYP46A1) in the Brain?

We previously described a heterozygous mouse model overexpressing human HA-tagged 24S-hydroxylase (CYP46A1) utilizing a ubiquitous expression vector. In this study, we generated homozygotes of these mice with circulating levels of 24OH 30–60% higher than the heterozygotes. Female homozygous CYP46A1 transgenic mice, aged 15 months, showed an improvement in spatial memory in the Morris water maze test as compared to the wild type mice. The levels of N-Methyl-D-Aspartate receptor 1, phosphorylated-N-Methyl-D-Aspartate receptor 2A, postsynaptic density 95, synapsin-1 and synapthophysin were significantly increased in the hippocampus of the CYP46A1 transgenic mice as compared to the controls. The levels of lanosterol in the brain of the CYP46A1 transgenic mice were significantly increased, consistent with a higher synthesis of cholesterol. Our results are discussed in relation to the hypothesis that the flux in the mevalonate pathway in the brain is of importance in cognitive functions.     

Measurement of phenyllactate, phenylacetate, and phenylpyruvate by negative ion chemical ionization-gas chromatography/mass spectrometry in brain of mouse genetic models of phenylketonuria and non-phenylketonuria hyperphenylalaninemia

Phenylketonuria (PKU) (OMIM 261600) is the first Mendelian disease to have an identified chemical cause of impaired cognitive development. The disease is accompanied by hyperphenylalaninemia (HPA) and elevated levels of phenylalanine metabolites (phenylacetate (PAA), phenyllactate (PLA), and phenylpyruvate (PPA)) in body fluids. Here we describe a method to determine the concentrations of PAA, PPA, and PLA in the brain of normal and mutant orthologous mice, the latter being models of human PKU and non-PKU HPA. Stable isotope dilution techniques are employed with the use of [(2)H(5)]-phenylacetic acid and [2,3, 3-(2)H(3)]-3-phenyllactic acid as internal standards. Negative ion chemical ionization (NICI)-GC/MS analyses are performed on the pentafluorobenzyl ester derivatives formed in situ in brain homogenates. Unstable PPA in the homogenate is reduced by NaB(2)H(4) to stable PLA, which is labeled with a single deuterium and discriminated from endogenous PLA in the mass spectrometer on that basis. The method demonstrates that these metabolites are easily measured in normal mouse brain and are elevated moderately in HPA mice and greatly in PKU mice. However, their concentrations are not sufficient in PKU to be "toxic"; phenylalanine itself remains the chemical candidate causing impaired cognitive development.     

Histamine Transmission Modulates the Phenotype of Murine Narcolepsy Caused by Orexin Neuron Deficiency

Narcolepsy type 1 is associated with loss of orexin neurons, sleep-wake derangements, cataplexy, and a wide spectrum of alterations in other physiological functions, including energy balance, cardiovascular, and respiratory control. It is unclear which narcolepsy signs are directly related to the lack of orexin neurons or are instead modulated by dysfunction of other neurotransmitter systems physiologically controlled by orexin neurons, such as the histamine system. To address this question, we tested whether some of narcolepsy signs would be detected in mice lacking histamine signaling (HDC-KO). Moreover, we studied double-mutant mice lacking both histamine signaling and orexin neurons (DM) to evaluate whether the absence of histamine signaling would modulate narcolepsy symptoms produced by orexin deficiency. Mice were instrumented with electrodes for recording the electroencephalogram and electromyogram and a telemetric arterial pressure transducer. Sleep attacks fragmenting wakefulness, cataplexy, excess rapid-eye-movement sleep (R) during the activity period, and enhanced increase of arterial pressure during R, which are hallmarks of narcolepsy in mice, did not occur in HDC-KO, whereas they were observed in DM mice. Thus, these narcolepsy signs are neither caused nor abrogated by the absence of histamine. Conversely, the lack of histamine produced obesity in HDC-KO and to a greater extent also in DM. Moreover, the regularity of breath duration during R was significantly increased in either HDC-KO or DM relative to that in congenic wild-type mice. Defects of histamine transmission may thus modulate the metabolic and respiratory phenotype of murine narcolepsy.     

肥胖合并高脂血症患者血清食欲素A、25-羟维生素D3、瘦素水平与胰岛素抵抗、脂代谢紊乱和肥胖评价指标的相关性分析

摘要 目的：探讨肥胖合并高脂血症患者血清食欲素A（orexin A）、25-羟维生素D3[25-(OH)D3]、瘦素（Leptin）水平与胰岛素抵抗、脂代谢紊乱和肥胖评价指标的相关性。方法：选择2019年2月至2021年12月中国医科大学附属第四医院收治的105例肥胖合并高脂血症患者为研究组,另取同期在中国医科大学附属第四医院健康体检的73例志愿者为对照组。检测并对比两组血清orexin A、25-(OH)D3、Leptin、胰岛素抵抗相关指标、脂代谢指标及肥胖评价指标水平的差异。采用Pearson相关性分析血清orexin A、25-(OH)D3、Leptin水平与胰岛素抵抗相关指标、脂代谢指标及肥胖评价指标的相关性。结果：研究组血清orexin A、25-(OH)D3水平低于对照组,而Leptin水平高于对照组（P＜0.05）。研究组空腹血糖（FPG）、空腹胰岛素（FINS）、胰岛素抵抗指数（HOMA-IR）水平均高于对照组（P＜0.05）。研究组总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）水平高于对照组,而高密度脂蛋白胆固醇（HDL-C）水平低于对照组（P＜0.05）。研究组体质量指数（BMI）、腰臀比、腰高比均高于对照组（P＜0.05）。肥胖合并高脂血症患者的血清orexin A、25-(OH)D3水平与FPG、FINS、HOMA-IR、TC、TG、LDL-C水平、BMI、腰臀比、腰高比均呈负相关,与HDL-C水平呈正相关（P＜0.05）;Leptin水平与FPG、FINS、HOMA-IR、TC、TG、LDL-C水平、BMI、腰臀比、腰高比均呈正相关,与HDL-C水平呈负相关（P＜0.05）。结论：肥胖合并高脂血症患者血清orexin A、25-(OH)D3水平降低,Leptin水平升高,且与胰岛素抵抗、脂代谢紊乱及肥胖指标升高有关。     

MAOB: a modifier gene in phenylketonuria?

Phenylketonuria (PKU), the most frequent inborn error of metabolism (1/15,000 live births), is an autosomal recessive condition caused by phenylalanine hydroxylase deficiency. Despite early and strict dietary control, some PKU children still exhibit behavioral and cognitive difficulties suggestive of a partly prenatal brain injury. The reported variability between the cognitive and clinical phenotypes within the same family raises the question of modifying genes in PKU. We suggest here that monoamine oxidase type B, MAOB, an enzyme degrading phenylethylamine, a very toxic metabolite of phenylalanine, could act as a modifying gene since a variant enzymatic activity of MAOB in PKU patients with similar phenylalanine levels would result in different phenylethylamine levels and different clinical outcomes. Finally the report of low MAOB, and consequently expectedly high phenylethylamine levels in neonates is consistent with a phenylethylamine-mediated brain injury possibly causing irreversible damages in PKU newborns prior to onset of the low protein diet.     

Orexin deficiency modulates cognitive flexibility in a sex-dependent manner

Cognitive flexibility is an important executive function and refers to the ability to adapt behaviors in response to changes in the environment. Of note, many brain disorders are associated with impairments in cognitive flexibility. Several classical neurotransmitter systems including dopamine, acetylcholine and noradrenaline are shown to be important for cognitive flexibility, however, there is not much known about the role of neuropeptides. The neuropeptide orexin, which is brain-widely released by neurons in the lateral hypothalamus, is a major player in maintaining sleep/wake cycle, feeding behavior, arousal, and motivational behavior. Recent studies showed a role of orexin in attention, cognition and stress-induced attenuation of cognitive flexibility by disrupting orexin signaling locally or systemically. However, it is not known so far whether brain-wide reduction or loss of orexin affects cognitive flexibility. We investigated this question by testing male and female orexin-deficient mice in the attentional set shifting task (ASST), an established paradigm of cognitive flexibility. We found that orexin deficiency impaired the intra-dimensional shift phase of the ASST selectively in female homozygous orexin-deficient mice and improved the first reversal learning phase selectively in male homozygous orexin-deficient mice. We also found that these orexin-mediated sex-based modulations of cognitive flexibility were not correlated with trait anxiety, narcoleptic episodes, and reward consumption. Our findings highlight a sexually dimorphic role of orexin in regulating cognitive flexibility and the need for further investigations of sex-specific functions of the orexin circuitry.     

Cholecystokinin levels in prohormone convertase 2 knock-out mouse brain regions reveal a complex phenotype of region-specific alterations

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.     

人载脂蛋白C3基因转基因小鼠的建立及血脂变化分析

目的制备系统性表达人载脂蛋白C3（APOC3）基因的转基因小鼠,建立高血脂小鼠模型。方法将人APOC3基因插入系统性表达启动子下游,构建转基因表达载体,通过显微注射法建立人APOC3转基因C57BL/6J小鼠。并利用特异引物PCR法鉴定转基因小鼠的基因型,Western blot检测基因表达水平,血生化分析检测不同月龄转基因小鼠与同龄野生型小鼠的血脂指标,脂肪染色观察肝脏脂肪水平。结果建立了高表达人APOC3基因的转基因小鼠品系;转入的人APOC3基因在血液、肝脏、小肠、肌肉、心脏、肾脏、脾脏中均有明显表达;不同月龄转基因小鼠的血浆甘油三酯水平明显高于同龄野生型小鼠;转基因小鼠的肝脏脂肪含量高于野生型小鼠。结论系统性表达人APOC3基因的转基因小鼠表现高脂血症表型,可以作为高血脂以及高血脂相关的心血管病的工具动物。     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Orexin neurons receive glycinergic innervations

Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.     

IL-17A对慢性阻塞性肺疾病的干预作用及其机制*

目的:探讨白细胞介素-17A(IL-17A)对慢性阻塞性肺疾病(COPD)的干预作用及其机制。方法:C57BL/6小鼠随机分为野生型空白对照组、野生型COPD组和IL-7A敲除COPD组,每组20只。野生型空白对照组小鼠不做任何处理,其余两组小鼠暴露于香烟烟雾(1支/次,4次/日,每次45 min,每次间隔时间为1 h,总干预时间为90 d)制作COPD模型。干预结束24 h后,利用动物肺功能检测系统测定小鼠肺功能。收集小鼠支气管肺泡灌洗液(BALF),测定BALF细胞计数和分类。收集小鼠肺组织,采用流式细胞法测定气道上皮IL-17A表达水平,采用酶联免疫吸附法测定肺组织炎症因子水平。采用蛋白免疫印迹法测定小鼠肺组织JNK/AP1信号通路蛋白表达水平。结果:与野生型空白对照组小鼠比较,野生型COPD组小鼠气道上皮IL-17A表达水平明显升高,吸气峰流速(PIF)和呼气峰流速(PEF)明显降低,BALF中性粒细胞、嗜酸性粒细胞、淋巴细胞和巨噬细胞数明显升高,肺组织CXC类趋化因子1(CXCL1)、CXC类趋化因子2(CXCL2)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)表达水平明显升高,JNK、cJun和cFos磷酸化水平及AP1表达水平明显升高(P＜0.05);与野生型COPD组小鼠比较,IL-7A敲除COPD组小鼠气道上皮IL-17A表达水平明显降低,PIF和PEF明显升高,BALF中性粒细胞、嗜酸性粒细胞、淋巴细胞和巨噬细胞数明显降低,肺组织CXCL1、CXCL2、IL-1β和IL-6表达水平明显降低,JNK、cJun和cFos磷酸化水平及AP1表达水平明显降低(P＜0.05)。结论:香烟烟雾可诱导小鼠气道上皮产生IL-17A,降低(或抑制)IL-17A的产生(或表达或分泌),通过抑制JNK/AP1信号通路,减轻COPD气道炎症反应,改善COPD小鼠肺功能。     

Role of Catalase and Superoxide Dismutase Activities on Oxidative Stress in the Brain of a Phenylketonuria Animal Model and the Effect of Lipoic Acid

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase which leads to accumulation of phenylalanine and its metabolites in tissues of patients with severe neurological involvement. Recently, many studies in animal models or patients have reported the role of oxidative stress in PKU. In the present work we studied the effect of lipoic acid against oxidative stress in rat brain provoked by an animal model of hyperphenylalaninemia (HPA), induced by repetitive injections of phenylalanine and α-methylphenylalanine (a phenylalanine hydroxylase inhibitor) for 7 days, on some oxidative stress parameters. Lipoic acid prevented alterations on catalase (CAT) and superoxide dismutase (SOD), and the oxidative damage of lipids, proteins, and DNA observed in HPA rats. In addition, lipoic acid diminished reactive species generation compared to HPA group which was positively correlated to SOD/CAT ratio. We also observed that in vitro Phe inhibited CAT activity while phenyllactic and phenylacetic acids stimulated superoxide dismutase activity. These results demonstrate the efficacy of lipoic acid to prevent oxidative stress induced by HPA model in rats. The possible benefits of lipoic acid administration to PKU patients should be considered.