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Acta Academiae Medicinae Sinicae

Abbreviation (ISO4): Acta Academiae Medicinae Sinicae      Editor in chief: Xuetao CAO

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Acta Academiae Medicinae Sinicae >

2017 , Vol. 39 >Issue 5: 705 - 714

DOI: https://doi.org/10.3881/j.issn.1000-503X.2017.05.018

Orginal Article

Emerging Roles of Histidine Triad Nucleotide Binding Protein 1 in Neuropsychiatric Diseases

  • DANG Yonghui , 1 ,
  • LIU Zhongwei 2 ,
  • LIU Peng 1 ,
  • WANG Jiabei 3
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  • 1Key Laboratory of the Health Ministry for Forensic Medicine,Key Laboratory of Environment and Genes Related to Diseases of the Education Ministry,College of Medicine & Forensics,Xi’an Jiaotong University Health Science Center,Xi’an 710061,China
  • 2Department of Cardiology,Shaanxi Provincial People’s Hospital,Xi’an 710061,China
  • 3Department of Pharmaceutical Sciences,School of Pharmacy,University of Maryland,Baltimore MD 21201,USA
Corresponding author:DANG Yonghui Tel:029-82655237,E-mail:

Received date: 2016-08-10

  Online published: 2017-10-30

Supported by

Supported by the National Natural Sciences Foundation of China(81171262,81771435) and the Natural Science Basic Research Plan in Shaanxi Province of China(2016JM8078)

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Abstract

The histidine triad nucleotide binding protein1(HINT1),which belongs to the histidine triad(HIT) enzyme superfamily,exerts its enzymic activities as hydrolase or transferase. Its physiological functions are still unclear. HINT1 protein is expressed in various tissues and plays an important role in transcription and signal transduction. Earlier studies have identified HINT1 as a haplo-insufficient tumor suppressor. Other evidences indicate that HINT1 is involved in a wide variety of physiological processes,some of which are irrelevant with its basic enzymic activities. Investigations recently suggest that HINT1 is closely related to many peripheral and central nervous system diseases,and plays a vital role in some of neuropsychiatric diseases such as inherited peripheral neuropathies,schizophrenia,mood disorder,drug addiction,and Down’s syndrome. In this review,the role of HINT1 in above-mentioned neuropsychiatric disorders was summarised,and the research findings of HINT1 in each of the above diseases were summarized and analyzed,in order to provide some guidance for further research on this protein.

Key words： histidine triad nucleotide binding protein 1; neuropathology

Cite this article

DANG Yonghui , LIU Zhongwei , LIU Peng , WANG Jiabei . Emerging Roles of Histidine Triad Nucleotide Binding Protein 1 in Neuropsychiatric Diseases[J]. Acta Academiae Medicinae Sinicae, 2017 , 39(5) : 705 -714 . DOI: 10.3881/j.issn.1000-503X.2017.05.018

三联组氨酸核苷结合蛋白(histidine triad nucleotide binding protein,HINT)是三联组氨酸(histidine triad,HIT)蛋白核苷酸转移酶和水解酶超家族成员,包含高度保守的活性位点基序His-X-His-X-His-XX(X代表疏水氨基酸)[1]。HINT亚家族十分古老,广泛表达于原核生物和真核生物,但其生物学功能尚未完全阐明。已经发现在得到全长测序的所有基因组中至少存在1个HINT,人类基因组包括3个独立的基因,分别编码HINT1、HINT2及HINT3基因产物。HINT2与HINT1有61%的序列一致性,而HINT3与HINT1仅有28%的序列一致[2]。
HINT1蛋白编码基因位于人类染色体5q31.2,其基因序列全长6160 bp,含有3个外显子,mRNA序列由782个碱基组成,编码产物是含126个氨基酸的细胞溶质蛋白,相对分子质量约14×103 [3-4]。
HINT1 1990年被首度发现是蛋白激酶抑制剂[5],在早期文献中一直被称作蛋白激酶C抑制剂-1(protein kinase C inhibitor-1,PKCI-1) [6-7]。尽管HINT1可能存在与蛋白激酶C(protein kinase C,PKC)的直接或间接交互作用,其PKC抑制作用现在被认为是存疑的[8],也正因为如此,PKCI-1被重新命名为HINT1[1]。但目前有文献依然沿用PKCI-1这一名称或是指Klein等[8]发现该蛋白能与PKC交互作用,遂将其命名为蛋白激酶C交互作用蛋白1(protein kinase C-interacting protein 1,PKCI 1),最近研究表明其中的PKCγ作为在神经元突触后膜的特异性蛋白,在HINT1敲除鼠的多个脑区高表达,但其活化却受到抑制[9]。根据结构研究,HINT1属嘌呤核苷酸结合蛋白[1,10],形成同源二聚体起作用,每一个亚单位结合1个核苷酸。
HINT1蛋白在多种组织中表达,在人类和啮齿类动物的肝、肾、脑、胃中均有分布[8]。HINT1蛋白广泛分布于小鼠中枢神经系统(central nervous system,CNS),尤其在嗅觉系统、大脑皮层、海马和部分丘脑、中脑、延髓中丰度较高[11]。

HINT1功能研究概况

HINT属于HIT酶家族的一个分支,主要包括HINT1、HINT2和HINT3,催化AMP-lysine、AMP-alanine、AMP-NH2 P-N键的水解过程,然而这一酶的生物学功能尚不清楚。
HINT2分布于肝脏、肾上腺皮质和胰腺等脏器,主要在细胞的线粒体内膜中,可能参与促进胆固醇由胞质向线粒体的转运,这一点与主要分布在细胞质的HINT1蛋白不同[12]。而HINT3与其他HINT蛋白不同,序列上的同源性较小,对酰基核苷酸底物有着特殊的亲和性。研究人员采用HINT1基因敲除(knockout,KO,HINT1-/-)小鼠研究HINT1在肿瘤发生中的作用,结果显示,在2~3岁时,KO小鼠自发性肿瘤发生率明显高于野生型(wild type,WT)小鼠[13-14]。在使用致癌剂诱导乳腺肿瘤发生时,HINT1-/-小鼠乳腺和卵巢肿瘤的发生率较之HINT1+/+小鼠显著增加;而HINT1-/-和HINT1+/-具有同样的肿瘤发生率,说明HINT1单等位基因表达似乎不足以抑制肿瘤,因此HINT1是单倍剂量不足肿瘤抑制因子[13]。HINT1对于氨基磷酸酯和酰基腺苷酸具有水解酶活性[15-16],但这一活性与其肿瘤抑制功能是否相关仍不清楚。
HINT1作为肿瘤抑制因子的作用机制并不完全清楚。最研究表明,HINT1抑制转录因子激活物蛋白-1 [17]、转录因子Ⅱ H[18]、小眼畸形转录因子[19-20]和上游刺激因子2[21]的活性,影响Wnt/beta-catenin通路上一些基因的转录过程[22]。因此,HINT1可能作为基因转录调节因子发挥重要的细胞功能,并进一步发挥肿瘤抑制蛋白的作用[23-24]。HINT1在人类特异性肿瘤中的表达缺陷与临床相关性是目前研究的热点之一。
HINT1广泛参与一系列生理过程,部分功能与其基本的酶活性无关[25]。除了肿瘤抑制功能,该蛋白还参与DNA损伤反应[13],调控细胞内钙离子信号传导[26],参与一些蛋白及转录因子的调控[9,24,27]。此外,还有证据表明细胞膜HINT1可经由PKC调控G蛋白偶联受体(G-protein-coupled receptor,GPCR)功能[28-32],调控谷氨酸N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受体与GPCR如μ阿片受体(μ-opioid receptor,MOR)和大麻素1型受体(cannabinoid receptors type 1,CNR1)的相互作用[33]。HINT1在细胞膜以同源二聚体形式存在,作为受氧化还原过程调控的支架蛋白而起作用,使各种通路受GPCR调控[34-35],并可能以这种方式调节HINT1蛋白的酶活性与其开启或关闭GPCR中介的不同信号通路的功能[36],如谷氨酸NMDA受体中介的突触可塑性、β-catenin调控、钙离子信号与DNA修复。尽管存在如此多研究,但我们仍缺乏对这些现象本质的认识。

HINT1的神经病理新功能

HINT1在神经系统中的生物学功能是新近的发现,我们新近的一项研究提示HINT1可能与多种神经精神疾病潜在相关[37],其他研究则表明这一分子参与多种神经精神疾病的发病。
精神分裂症 HINT1基因位于5q31.2遗传位点内,这一区域与精神分裂症关联[38-39]。精神分裂症是一种临床常见但病因不是十分清楚的精神疾病,临床表现包括幻觉、妄想、认知障碍、情感及行为异常等。这一疾病的病因极其复杂,包括遗传易感性与表观遗传和环境因素的交互作用[40]。
Vawter等[41-42]研究发现,精神分裂症患者背外侧前额皮质(dorsolateral prefrontal cortex,DLPFC)内的HINT1 mRNA低水平表达,他们随后采用RT-PCR和原位杂交方法进一步证实了上述结论[43]。Chen等[44]在一项对染色体5q22-33区域进行的精细定位研究中发现,SPEC2/PDZ-GEF2/ACSL6区域单倍型与精神分裂症关联,而HINT1基因位于这一区域中。此后,他们对爱尔兰精神分裂症高发家族研究(Irish study of high density schizophrenia families,ISHDSF;含1350个受试者及273个家系)及爱尔兰精神分裂症病例—对照研究(Irish case-control study of schizophrenia,ICCSS;含655受累个体和626例对照)样本HINT1基因周围区域8个SNPs进行鉴定,又通过斯坦利医学研究所提供的精神分裂症患者和健康对照者尸脑样本cDNA对比HINT1表达水平,发现HINT1的变异可能与精神分裂症关联[3]。Varadarajulu等[45]比较了精神分裂症患者与健康对照者死后DLPFC和丘脑的HINT1蛋白表达,发现HINT1在DLPFC下调,在丘脑上调,2011年一项研究也有类似发现[46]。上述研究还发现HINT1与精神分裂症的关联存在性别特异性,可能仅存在于男性患者[3,43-44]。
上述病例研究结果得到了HINT1 KO小鼠研究结果的支持。美国马里兰大学的研究人员发现,与WT对照相比,HINT1 KO小鼠自主活动减少;急性给予苯丙胺(amphetamine,AMPH)可显著增加WT小鼠自主活动,但KO小鼠对此的反应更加明显;定量微透析研究结果表明,KO小鼠纹状体或者伏核(nucleus accumbens,NAc)内多巴胺(dopamine,DA)动力学没有发生改变,AMPH急性增加DA水平的能力也没有改变,这说明KO小鼠行为的改变与这些区域突触前DA神经传递功能无关。该研究进一步发现与WT小鼠相比,系统性给予多巴胺受体直接激动剂阿扑吗啡也可显著增加KO小鼠的自主活动,提示KO小鼠突触后的DA功能发生了改变[47]。上述结果证实,HINT1在调节AMPH行为反应中发挥重要作用,其缺失可能导致突触后DA传递功能紊乱。考虑到精神分裂症多巴胺能神经系统功能亢进的发病假说[48],与此同时在精神分裂样动物模型中,AMPH诱发的高活动性被视为啮齿类动物精神分裂症阳性症状样行为改变,并与精神分裂症阳性症状的皮层下多巴胺功能亢进相关[49],因此HINT1 KO小鼠似乎可作为精神分裂症的遗传性动物模型。
周围神经病 遗传性运动感觉性周围神经病(hereditary motor and sensory neuropathy,HMSN)是累及外周神经系统(peripheral nervous system,PNS)的神经肌肉和神经变性性障碍,干扰脑与躯体的联系。它们是最常见到的遗传性神经肌肉障碍,大约2500个个体中就有1个发病[50]。该病分为7型(Ⅰ~Ⅶ),其中Ⅰ、Ⅱ两型又称为腓骨肌萎缩症(charcot-marie-tooth disease,CMT)[51]。HMSN囊括一大组涉及多个基因和复杂表型的疾病,因此正确诊断各遗传亚型对于临床医生来说是一个棘手的问题。目前已鉴定出超过100种不同亚型的HMSN,每一种均有其特异的临床特征、病理生理及预后。不同的临床特征和发展模式已被用于区分特异的遗传亚型,指导分子筛选并提供合理的遗传咨询。然而直到最近,仍有许多病例未明确突变,因而无法进行分子诊断。近年来对CMT的临床研究发现了诸多与该病相关的基因,其中就包括HINT1[52-54]。有案例报道表明,HINT1突变有其特殊的症状表型[55]。
Zimon等[56]对50个具有神经性肌强直(neuromyotonia,NM)的常染色体隐性轴突性神经病(autosomal recessive axonal neuropathy,ARAN)患者进行鉴定,结果发现8个不同的HINT1突变。NM是一种罕见的神经肌肉障碍,通常发生于儿童晚期到成年早期。这一障碍以由PNS运动轴突过度兴奋引起肌肉自主收缩后迟发性松弛为特征[57]。Zimon等[56]详细记录了神经传导、同心针肌电图及肌肉/神经活检等临床特征,为了确证HINT1与具有NM的ARAN之间的连锁关系,又对262个散发的具有常染色体隐性遗传外周神经病的无关患者进行了HINT1突变筛选,结果发现HINT1突变的频率为11%,而在ARAN-NM患者则增至76%,表现出非常强的因果性遗传关联。
Zimon等[56]还分析了HINT1在成年小鼠不同组织(包括心脏、肌肉、脑、脊髓、坐骨神经、脾脏、肝脏和肺脏)中的表达,结果发现HINT1蛋白在小鼠坐骨神经中高表达,这说明HINT1是PNS功能的重要组分。他们继而进行了功能性确证实验,其结果进一步表明HINT1突变属功能丧失性突变。因此,HINT1功能丧失性突变属常染色体隐性遗传神经病新基因,决定了一种新遗传亚型,即ARAN-NM。然而,HINT1在遗传性周围神经病变病理生理中的角色依然未知,猜测可能与PNS毒性代谢产物的蓄积有关[56]。Zimon等[58]的最新工作通过对包括HINT1在内的一些CMT基因进行筛选分析,可以完成对 41.3%CMT患者的分子诊断。
然而,Seburn等[59]检测了HINT1 KO小鼠,发现与WT小鼠相比,KO小鼠虽然在旷场实验中移动较慢,活动时间减少,但其运动相关测试结果未见异常;其肌肉、神经肌接头及郎飞结结构正常,无变性或再生迹象;4~13月龄时外周神经轴突数和髓鞘形成正常;4月龄时虽然轴突略小,但传导速度并不减少;13月龄时轴突直径无差异;在使用非生理刺激或应激源后,如降低温度、阻滞钾通道,肌电图无法记录到神经肌强直。作者据此认为HINT1 KO小鼠可能对研究HINT1蛋白的生物化学活性有效,但对HINT1相关神经病和神经肌强直则不能认为是一种动物模型。
此外,Horga等[60]通过对152例英国及西班牙遗传性神经病患者的直接测序并未发现HINT1基因的变异,这表明HINT1与该病的关联关系存在地域特异性[60-62]。还有研究表明HINT1突变也会导致远端遗传性运动神经病(distal hereditary motor neuropathies,dHMNs)[53]。
心境障碍 心境障碍是以显著而持久的情感高涨(躁狂)或低落(抑郁)为主要特征的一组精神障碍,临床上主要表现有抑郁症(major depressive disorder,MDD)和双相障碍(bipolar disorder,BP)。
Elashoff等[63]对12篇采用微阵列技术研究双相障碍的文献进行了Meta分析,结果发现双相障碍患者脑组织中HINT1表达下降。对HINT1 KO小鼠的研究则发现:与WT对照相比,KO小鼠的抗抑郁样行为增多;急性给予300 mg/kg心境稳定剂丙戊酸盐(valproic acid,VPA)可缓解KO小鼠在悬尾实验(tail suspension test,TST)中的活动异常;在水迷宫定位导航任务中,KO小鼠表现出学习与记忆能力增强[64];而HINT1 KO小鼠下午血浆皮质酮水平显著高于WT。HINT1 KO小鼠表现出的行为和内分泌表型说明HINT1缺乏会导致情绪功能发生改变,产生抗抑郁样行为及血浆皮质酮升高,这就是说,HINT1基因在CNS中参与情绪调控,HINT1的缺乏可能导致躁狂样行为。
与此相对应的是,Martins-de-Souza等[65]采用蛋白质组学方法分析24例MDD患者和12例匹配对照者死后DLPFC脑组织,继而采用Wetern blot法或质谱法确证差异性蛋白表达,结果发现MDD患者HINT1表达增加,且只在无精神病性症状的MDD增加。Ge等[66]对大鼠慢性温和应激(chronic mild stress,CMS)抑郁模型海马进行分析,结果发现HINT1水平比对照组大鼠高,而经潜在抗抑郁剂长链脂肪酸酰胺油酰胺干预后HINT1并不降低,据此作者推测HINT1可能是MDD的生物标志物。
焦虑障碍 HINT1与焦虑障碍的关系未见临床相关研究报道,主要集中于基因敲除小鼠的研究。
Barbier等[47]研究发现,与WT对照相比,HINT1 KO雄性小鼠自主活动减少;在水迷宫中表现出较少趋触性;在明暗箱测试期间KO小鼠焦虑样行为减少,也就是说HINT1缺乏所致情绪行为发生改变包括抗焦虑样行为[64]。
然而,Varadarajulu等[67]对雄性HINT1 KO小鼠进行了一系列行为测试后发现,与WT小鼠相比,HINT1 KO雄性小鼠自主活动及普通探索活动无显著性差异;在高架十字迷宫及明暗箱测试中,HINT1 KO小鼠较其同窝WT小鼠表现出更多的焦虑样行为。
Jackson等[68]发现尼古丁急性给药并不对HINT1 KO雄鼠产生抗焦虑作用,而是产生致焦虑反应,安定也不能诱导这些小鼠的抗焦虑反应,说明HINT1 KO雄鼠存在广泛性焦虑的表型;上述焦虑样行为并未在雌鼠中得到观察,这进一步支持HINT1的影响具有性别差异。
如上所述,利用基因敲除小鼠进行焦虑样行为结果不尽一致,推测可能与在实验方法、实验装置、动物年龄(尤其是动物年龄,Barbier等[47]采用的动物年龄较大)等方面存在的差异有关。
疼痛与镇痛 人类μ阿片受体属于GPCR,是吗啡镇痛和阿片成瘾的重要分子靶点。Guang等[32]首次发现HINT1与人类MOR受体C末端特异性的交互作用减少了MOR的脱敏和磷酸化;考虑到MOR的磷酸化和脱敏在吗啡耐受中发挥重要作用,他们进行了吗啡慢性给药后热板实验,结果表明在HINT1 KO小鼠基础痛阈升高,同时吗啡诱导的镇痛效应显著增强,并且KO小鼠表现出对吗啡镇痛的较大程度的耐受,这说明HINT1在MOR中介的吗啡镇痛中起负性调节作用[32]。Jackson等[68]发现雄性HINT1 KO小鼠在甩尾测试而非热板测试中,对急性给予尼古丁引起的抗伤害感受效应缺乏敏感性。我们对HINT1 KO鼠的研究则发现,在基础状态下,HINT1 KO小鼠在热板、机械刺激痛测试及福尔马林炎性痛测试中痛觉感受均表现出更高的敏感性,此外,并未发现有性别差异[69]。有研究人员发现了HINT1蛋白稳定MOR受体、CNR1等GPCR和谷氨酸亲离子受体N(GLuN,即NMDA)的关联,这种偶联对吗啡的镇痛效力是必需的,在神经病理性疼痛发生时,GLuN过度激活,MOR受体、CNR1与GLuN的关联减弱[70-71]。Garzon等[72]运用HINT1酶活性抑制剂鸟苷-5’-色胺氨基甲酸酯(guanosine-5’-tryptamine carbamate,TpGc)的研究发现,TpGc显著提高了吗啡的镇痛作用,防止了耐受的形成。在分子层面,TpGc降低了MORS募集NMDAR活动及负性调节阿片信号传导的能力。小鼠经历长期神经压榨损伤伴随着NMDA受体活性增加,单次脑室内给予TpGc可减弱NMDA受体功能,减轻机械性异常疼痛(触诱发痛)。这表明,HINT1抑制剂可在急性和神经病理性疼痛的临床治疗中发挥潜在的作用。然而,一项对2294位欧洲癌痛患者的关联研究未能发现HINT1基因的SNP变异与阿片类药物使用剂量存在显著关联[73]。
药物成瘾 来自两个独立样本的关联分析表明,HINT1基因变异与尼古丁依赖的表型关联,进一步的人类死亡后mRNA表达分析表明吸烟状态与表型和HINT1脑内表达水平有关;动物研究中,Western blot分析表明慢性尼古丁给药后小鼠的NAc内HINT1蛋白水平增加,而在给予尼古丁拮抗剂美卡拉明,或者停止给予尼古丁24和72 h后,HINT1增加幅度减少[74]。Fang等[75]对284例目前或继往吸烟的中国人群的SNP分析发现,HINT1 rs3853309与吸烟状态相关。上述研究结果表明HINT1和尼古丁依赖存在遗传关联,尼古丁对HINT1水平的调节可能参与尼古丁成瘾过程。然而在一项包括374位尼古丁依赖吸烟者的开放式随机试验中,他们接受尼古丁替代治疗(nicotine replacement therapy,NRT),实验结果并不支持HINT1基因变异与戒烟成功间存在关联[76]。
Jackson等[77]对雄性HINT1 KO和WT小鼠进行了尼古丁条件位置偏爱(conditioned place preference,CPP)实验,评估了尼古丁戒断的躯体和情绪症状,并对尼古丁戒断后条件性位置厌恶(conditioned place aversion,CPA)的形成进行了测定,结果发现尼古丁不能使HINT1 KO小鼠产生显著性CPP,HINT1 KO小鼠尼古丁戒断时痛觉过敏和躯体症状较轻,表现出与WT对照相似的显著性尼古丁戒断性CPA。这一研究进一步支持HINT1基因在调节与尼古丁奖赏和尼古丁躯体戒断相关行为中发挥作用。
HINT1与其他成瘾性药物的关系研究较少。Romanova等[78]利用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)技术发现在单次注射10 mg/kg可卡因后,旷场实验中可卡因低反应大鼠(low cocaine responders,LCRs)内侧前额皮质(medial prefrontal cortex,mPFC)中HINT1峰强度增加。而已有的研究表明LCRs较之高反应者(high cocaine responders,HCRs)对可卡因诱导的行为敏化更敏感[79-80],表现出对可卡因奖赏效应的CPP增加[81],对可卡因自我给药动机增强[82]。这就是说,HINT1在可卡因成瘾的易感表型LCRs中表达升高。
脑衰老 衰老是阿尔茨海默病(Alzheimer’s disease,AD)等神经变性障碍的主要高危因素。然而,脑衰老过程中的分子变化极端复杂,并且大部分尚不清楚。Abdel Rassoul等[83]对哺乳动物倭狐猴颞叶皮层转录组进行了研究,鉴定出695个基因在老年动物、AD样动物与年轻成年动物皮层间存在差异。这些基因大约1/3在健康衰老动物和AD样动物中表现出相同的表达变化——其中包括HINT1和HINT2基因的下调,而超出2/3的基因在这两组动物中与年轻健康动物相比表达改变相反。结合前述2~3岁KO小鼠自发性肿瘤发生率比WT小鼠高得多,以及焦虑样行为在不同年龄KO小鼠可能表现不一的结果,可以猜测HINT1可能参与脑衰老过程,但可能不参与AD发病过程。
唐氏综合征 Weitzdoerfer等[84]采用蛋白质组学技术测定人类流产胎儿皮层组织,结果发现,出生前参与神经分化、迁移和突触传递的早期生命蛋白在唐氏综合征患者皮层存在缺陷,其中包括HINT1。

小 结

自从发现HINT1蛋白参与各种生物的多种生命现象以来,人们对于其研究兴趣一直有增无减。尽管这一领域已经有许多研究成果发表,HINT1在细胞生理中的真实功能依然不得而知。HIT蛋白在进化中较为保守,这说明HINT1行使着基本而重要的生理功能。HINT1神经病理功能是一个新兴的研究领域。
一方面,初步研究表明HINT1广泛参与各种神经系统的病理生理过程,继续深入研究HINT1酶活性、肿瘤抑制、神经病理之间的内在联系是一个突出的科学问题。例如,HINT1可发挥酶活性使5’-O-单硫代磷酸酯发生脱硫作用,在细胞内产生游离的硫化氢(H2S) [85];而H2S作为重要的气体信号分子,在肿瘤、精神系统疾病及心血管疾病等中均发挥重要作用[86-87]。总之,HINT1在转录及信号转导中的作用,可能会有助于揭示HINT1本质的细胞生理功能。
另一方面,HINT1与GPCR、NMDA之间的交互作用关系则是一个极有希望的对所有本文所涉及神经精神疾病的治疗具有启示意义的重要研究方向[88]。例如,CNR1受体C末端序列及NMDA受体NR1亚基C1节段均与HINT1蛋白二聚体直接相互作用,HINT1起加强二者联系的作用[88-89],HINT1缺乏时,NMDA受体不能与CB1建立功能性的交互作用,大麻类不能减少NMDA受体中介的钙流量、NO产物产生以及钙和锌从内部储存释放[34]。这一机制可解释大麻滥用产生NMDAR功能减退,从而引起精神病性症状或精神分裂症复发的临床现象。并且,NMDA受体功能不足新近被认为引起了精神分裂症患者纹状体和前额区的多巴胺能功能失调[90],这使得这一视角更具吸引力。
相信随着更多采用基因工程小鼠的实验开展,HINT1对神经病理和肿瘤发生的作用将会逐渐被阐明。

The authors have declared that no competing interests exist.

References

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[1]
Brenner C,Garrison P,Gilmour J,et al.Crystal structures of HINT demonstrate that histidine triad proteins are GalT-related nucleotide-binding proteins[J].Nat Struct Biol,1997,4(3):231-238.Histidine triad nucleotide-binding protein (HINT), a dimeric purine nucleotide-binding protein from rabbit heart, is a member of the HIT (histidine triad) superfamily which includes HINT homologues and FHIT (HIT protein encoded at the chromosome 3 fragile site) homologues. Crystal structures of HINT-nucleotide complexes demonstrate that the most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Galactose-1-phosphate uridylyltransferase, whose deficiency causes galactosemia, contains tandem HINT domains with the same fold and mode of nucleotide binding as HINT despite having no overall sequence similarity. Features of FHIT, a diadenosine polyphosphate hydrolase and candidate tumour suppressor, are predicted from HINT-nucleotide structures.

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Brenner C. Hint,Fhit,and GalT:function,structure,evolution,and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases[J].Biochemistry,2002,41(29):9003-9014.HIT (histidine triad) proteins, named for a motif related to the sequence HphiHphiHphiphi (phi, a hydrophobic amino acid), are a superfamily of nucleotide hydrolases and transferases, which act on the alpha-phosphate of ribonucleotides, and contain a approximately 30 kDa domain that is typically either a homodimer of approximately 15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution, and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5'-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases, including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylyl sulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in the development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding site. The potential roles of ASW and Hint in avian sexual development are discussed elsewhere. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases.

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Pearson JD,DeWald DB,Mathews WR,et al. Amino acid sequence and characterization of a protein inhibitor of protein kinase C[J].J Biol Chem,1990,265(8):4583-4591.The complete primary structure has been determined for an inhibitor protein of protein kinase C. The bovine brain-derived inhibitor has a pI of 6 and its N-terminal alanine residue is blocked by acetylation. Fragments obtained by chemical and enzymatic cleavage of the purified inhibitor were analyzed by Edman degradation, fast atom bombardment mass spectrometry, and tandem mass spectrometry. The results establish that the protein has a calculated average molecular mass of 13,690 daltons and contains 125 amino acid residues with the following sequence: (sequence: see text) The inhibitor does not show significant homology with any other known protein. Circular dichroism of the freshly prepared apoprotein indicated a secondary structural content of 23% alpha-helix, 31% beta-sheet, and 11% beta-turn. Immobilization on nitrocellulose followed by exposure to a 65Zn2(+)-containing overlay solution showed that, like protein kinase C itself, the inhibitor is a zinc-binding protein, although the sequence does not reveal a "zinc finger" structure. Competition with 10-fold molar excess Ca2+ or Mg2+ did not reduce the zinc-binding specificity of this inhibitor.

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Martin J,St-Pierre MV,Dufour JF.Hit proteins,mitochondria and cancer[J].Biochim Biophys Acta,2011,1807(6):626-632.doi:10.1016/j.bbabio.2011.02.001.Abstract The histidine triad (HIT) superfamily comprises proteins that share the histidine triad motif, His-0301-His-0301-His-0301-0301, where 0301 is a hydrophobic amino acid. HIT proteins are ubiquitous in prokaryotes and eukaryotes. HIT proteins bind nucleotides and exert dinucleotidyl hydrolase, nucleotidylyl transferase or phosphoramidate hydrolase enzymatic activity. In humans, 5 families of HIT proteins are recognized. The accumulated epidemiological and experimental evidence indicates that two branches of the superfamily, the HINT (Histidine Triad Nucleotide Binding) members and FHIT (Fragile Histidine Triad), have tumor suppressor properties but a conclusive physiological role can still not be assigned to these proteins. Aprataxin forms another discrete branch of the HIT superfamily, is implicated in DNA repair mechanisms and unlike the HINT and FHIT members, a defective protein can be conclusively linked to a disease, ataxia with oculomotor apraxia type 1. The scavenger mRNA decapping enzyme, DcpS, forms a fourth branch of the HIT superfamily. Finally, the GalT enzymes, which exert specific nucleoside monophosphate transferase activity, form a fifth branch that is not implicated in tumorigenesis. The molecular mechanisms by which the HINT and FHIT proteins participate in bioenergetics of cancer are just beginning to be unraveled. Their purported actions as tumor suppressors are highlighted in this review. Copyright 0008 2011 Elsevier B.V. All rights reserved.

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[7]
Huebner K,Saldivar JC,Sun J,et al.Hits,Fhits and Nits:beyond enzymatic function[J].Adv Enzyme Regul,2011,51(1):208-217.doi:10.1016/j.advenzreg.2010.09.003.We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion genewas discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5'-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5'-end of the fly Fhit gene, this gene was hailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have turned out to be at least as mysterious as the Fhit proteins, with the Nit1 substrate still unknown and the surprising finding that Nit proteins also appear to behave as tumor suppressor proteins. Whether the predicted enzymatic functions of these proteins are relevant to the observed biological functions, remain among the outstanding unanswered puzzles and raise the question: have these mammalian proteins evolved beyond the putative original enzymatic purpose, such that the catalytic function is now vestigial and subservient to signal pathways that use the protein-substrate complexes in pathways that signal apoptosis or DNA damage response? Or can these proteins be fulfilling catalytic functions independently but in parallel with signal pathway functions, as perhaps observed for Aprataxin? Or is the catalytic function indeed part of the observed biological functions, such as apoptosis and tumor suppression? Perhaps the recent, post-genomic focus on metabolomics and genome-wide investigations of signal pathway networks will lead to answers to some of these outstanding questions.

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Klein MG,Yao Y,Slosberg ED,et al.Characterization of PKCI and comparative studies with FHIT,related members of the HIT protein family[J].Exp Cell Res,1998,244(1):26-32.doi:10.1006/excr.1998.4153.We previously described the isolation of a human cDNA that encodes a protein termed protein kinase C inhibitor (hPKCI). We elucidated the three-dimensional structure of this protein and demonstrated that in vitro, it enzymatically hydrolyzes adenosine polyphosphates. To identify other proteins that interact with hPKCI, in the present study, we used the hPKCI as a bait in the yeast two-hybrid system, together with a mouse embryo cDNA library. This led to the isolation of a murine PKCI homologue (mPKCI). This finding is consistent with our previous structural studies indicating that hPKCI exists as a homodimer and indicates the strong conservation of the PKCI sequence during evolution. Northern blot analysis indicated that a 0.7-kb PKCI mRNA was expressed in several tissues obtained from adult mice and also in a variety of rodent and human cell lines. Western blot analyses, using a polyclonal antibody prepared against hPKCI, indicated that this protein is expressed at relatively high levels in several murine tissues and in a variety of human cell lines prepared from normal tissues or tumors. In contrast to these findings, parallel studies with a polyclonal antibody to FHIT, a related histidine triad (HIT) protein and putative tumor suppressor, indicated that FHIT was expressed at low or undetectable levels in some of the same cell lines. Microscopy of immunostained cells indicated that the PKCI protein was present mainly in the nucleus of both normal and tumor-derived epithelial cell lines. Evidence presented in this and previous studies suggest that in vivo the ubiquitously expressed PKCI protein does not function as an inhibitor of PKC but rather acts as an enzyme in a yet to be identified pathway.

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[9]
Zhang F,Fang Z,Wang JB.Hint1 knockout results in a compromised activation of protein kinase C gamma in the brain[J].Brain Res,2015,1622:196-203.doi:10.1016/j.brainres.2015.06.029.Abstract Previous studies have implicated a role of the histidine triad nucleotide-binding protein 1 (Hint1) in the pathogenesis of schizophrenia. Protein kinase C gamma (PKC0206) could be potentially involved in the Hint1-implicated pathogenesis since PKC0206 was identified as a Hint1 interacting protein. Recently, a debate was brought forward from the understanding how Hint1 affects the expression and activity of PKC0206 in the brain. In the present study, we use Hint1 knockout mice and biochemical analysis to define the effect of Hint1 on protein PKC0206. Our data reveal that Hint1-deficiency in mouse brains led to increased protein levels of PKC0206 in the cortex and hippocampus, the striatum and thalamus and amygdala. Without stimulation, PKC0206 protein in Hint1-deficient brain displayed a basal activity that was reflected by control-leveled phosphorylations of PKC0206 T514 and T674 at its kinase domain. Upon psycho-stimulation, both sites of PKC0206 T514 and T674 were activated in these brain structures via phosphorylation; however, the phosphorylation level at the site of PKC0206 T674 apparently attenuated in Hint1-deficient mice compared to wild-type control. Thus, we conclude that Hint1 deficiency leads to an increased protein level of PKC0206 in the brain and a compromised activation response of PKC0206 upon stimulation. These findings suggest an inhibitory role of Hint1 on the protein PKC0206 in the brain and an impaired PKC0206-mediated phosphorylation signal in Hint1-deficient neuron. Copyright 0008 2015 Elsevier B.V. All rights reserved.

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[10]
Bai G,Feng B,Wang JB,et al.Studies on ligand binding to histidine triad nucleotide binding protein 1[J].Bioorg Med Chem,2010,18(18):6756-6762. doi:10.1016/j.bmc.2010.07.051.Histidine triad nucleotide binding protein (HINT1) is an intracellular protein that binds purine mononucleotides. Strong sequence conservation suggests that these proteins play a fundamental role in cell biology, however its exact cellular function continues to remain elusive. nuclear magnetic resonance (NMR) studies using STD and HSQC were conducted to observe ligand binding to HINT1. These studies were confirmed using fluorescence spectroscopy titrations. We found that AICAR, the first non-phosphate containing ligand, binds to mouse histidine triad nucleotide binding protein 1 (HINT1). Chemical shift perturbations are mapped onto the X-ray structure showing AICAR binds at the same site as GMP. The NMR results demonstrated that this method will be valuable for the future screening of small molecules that can be used to modulate the function of HINT1.

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[11]
Liu Q,Puche AC,Wang JB.Distribution and expression of protein kinase C interactive protein(PKCI/HINT1) in mouse central nervous system(CNS)[J].Neurochem Res,2008,33(7):1263-1276.doi:10.1007/s11064-007-9578-4.

[12]
Lenglet S,Antigny F,Vetterli L,et al.Hint2 is expressed in the mitochondria of H295R cells and is involved in steroidogenesis[J].Endocrinology,2008,149(11):5461-5469.doi:10.1210/en.2008-0400.Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.

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[13]
Li H,Zhang Y,Su T,et al.Hint1 is a haplo-insufficient tumor suppressor in mice[J].Oncogene,2006,25(5):713-721.doi:10.1038/sj.onc.1209111.The HINT1 protein, a member of the histidine triad (HIT) family, is highly conserved in diverse species and ubiquitously expressed in mammalian tissues. However, its precise function in mammalian cells is not known. As a result of its structural similarity to the tumor-suppressor protein FHIT, we used homozygous-deleted Hint1 mice to study its role in tumorigenesis. We discovered that after 2 to 3 years of age the spontaneous tumor incidence in Hint1 -/- mice was significantly greater than that in wild-type Hint1 +/+ mice (P < 0.05). Using a well-established mouse model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis we found a marked and significant (P < 0.05) increase in the incidence of mammary and ovarian tumors in both, Hint1 -/- and +/- mice versus +/+ mice. The Hint1 -/- and +/- mice had similar tumor incidence and similar tumor histologies. Therefore, deletion of Hint1 in mice enhances both spontaneous tumor development and susceptibility to tumor induction by DMBA. In addition, since the Hint1 +/- tumors retained expression of the unmutated wild-type allele, Hint1 is haplo-insufficient with respect to tumor suppression in this model system.

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[14]
Su T,Suzui M,Wang L,et al.Deletion of histidine triad nucleotide-binding protein 1/PKC-interacting protein in mice enhances cell growth and carcinogenesis[J].Proc Natl Acad Sci USA,2003,100(13):7824-7829.doi:10.1073/pnas.1332160100.

[15]
Bieganowski P,Garrison PN,Hodawadekar SC,et al.Adenosine monophosphoramidase activity of Hint and Hnt1 supports function of Kin28,Ccl1,and Tfb3[J].J Biol Chem,2002,277(13):10852-10860.doi:10.1074/jbc.M111480200.Abstract The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhit-related hydrolases, and a branch of galactose-1-phosphate uridylyltransferase (GalT)-related transferases. Although substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit, and GalT are known, good substrates had not been reported for any member of the Hint branch, and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily. Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5'-monophosphoramidate (AMPNH(2)) in an active-site-dependent manner at second order rates exceeding 1,000,000 m(-1) s(-1). Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperatures. Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3, and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH and to mutations in Cak1, which phosphorylates Kin28. The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers. Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity.

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[16]
Chou TF,Baraniak J,Kaczmarek R,et al.Phosphoramidate pronucleotides:a comparison of the phosphoramidase substrate specificity of human and Escherichia coli histidine triad nucleotide binding proteins[J].Mol Pharm,2007,4(2):208-217.doi:10.1021/mp060070y.Abstract To facilitate the delivery of nucleotide-based therapeutics to cells and tissues, a variety of pronucleotide approaches have been developed. Our laboratory and others have demonstrated that nucleoside phosphoramidates can be activated intracellularly to the corresponding 5'-monophosphate nucleotide and that histidine triad nucleotide binding proteins (Hints) are potentially responsible for their bioactivation. Hints are conserved and ubiquitous enzymes that hydrolyze phosphoramidate bonds between nucleoside 5'-monophosphate and an amine leaving group. On the basis of the ability of nucleosides to quench the fluorescence of covalently linked amines containing indole, a sensitive, continuous fluorescence-based assay was developed. A series of substrates linking the naturally fluorogenic indole derivatives to nucleoside 5'-monophosphates were synthesized, and their steady state kinetic parameters of hydrolysis by human Hint1 and Escherichia coli hinT were evaluated. To characterize the elemental and stereochemical effect on the reaction, two P-diastereoisomers of adenosine or guanosine phosphoramidothioates were synthesized and studied to reveal a 15-200-fold decrease in the specificity constant (kcat/Km) when the phosphoryl oxygen is replaced with sulfur. While a stereochemical preference was not observed for E. coli hinT, hHint1 exhibited a 300-fold preference for d-tryptophan phosphoramidates over l-isomers. The most efficient substrates evaluated to date are those that contain the less sterically hindering amine leaving group, tryptamine, with kcat and Km values comparable to those found for adenosine kinase. The apparent second-order rate constants (kcat/Km) for adenosine tryptamine phosphoramidate monoester were found to be 107 M-1 s-1 for hHint1 and 106 M-1 s-1 for E. coli hinT. Both the human and E. coli enzymes preferred purine over pyrimidine analogues. Consistent with observed hydrogen bonding between the 2'-OH group of adenosine monophosphate and the active site residue, Asp43, the second-order rate constant (kcat/Km) for thymidine tryptamine phosphoramidate was found to be 3-4 orders of magnitude smaller than that for uridine tryptamine phosphoramidate for hHint1 and 2 orders of magnitude smaller than that for E. coli hinT. Ara-A tryptamine phosphoramidate was, however, shown to be a good substrate with a specificity constant (kcat/Km) only 10-fold lower than the value for adenosine tryptamine phosphoramidate. Consequently, nucleoside phosphoramidates containing unhindered primary amines and either an alpha or beta 2'-OH group should be easily bioactivated by Hints with efficiencies rivaling those for the 5'-monophosphorylation of nucleosides by nucleoside kinases. The differential substrate specificity observed for human and E. coli enzymes represents a potential therapeutic rationale for the development of selective antibiotic phosphoramidate pronucleotides.

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[17]
Wang L,Zhang Y,Li H,et al.Hint1 inhibits growth and activator protein-1 activity in human colon cancer cells[J].Cancer Res,2007,67(10):4700-4708.doi:10.1158/0008-5472.can-06-4645.Abstract There is accumulating evidence that histidine triad (HIT) nucleotide-binding protein 1 (HINT1), a member of the evolutionary highly conserved HIT protein super family, is a novel tumor suppressor. However, the mechanism of action of HINT1 with respect to tumor suppression is not known. In the present study, we found that a series of human colon cancer cell lines displayed various levels of expression of HINT1, with a very low level in SW480 cells. This cell line also displayed partial methylation of the promoter region of the Hint1 gene, and treatment of these cells with 5-azadeoxycitidine increased expression of Hint1 mRNA and protein. Therefore, the decreased expression of HINT1 in SW480 cells seems to be due to epigenetic silencing. Increased expression of HINT1 in these cells, using a retrovirus vector (pLNCX2) that encodes either wild-type (WT) Hint1 or a point mutant (His(112)/Asn(112)) of Hint1, inhibited the proliferation of SW480 cells. Because of the important role of the activator protein-1 (AP-1) transcription factor in cancer cells, we examined possible effects of HINT1 on AP-1 transcription factor activity in SW480 cells transfected with an AP-1-luciferase reporter. We found that cotransfection with a pHA-Hint1 plasmid DNA significantly inhibited this activity. Studies with inhibitors indicated that AP-1 activity in SW480 cells requires the activity of c-Jun NH(2)-terminal kinase (JNK) 2 and not JNK1. Cotransfection with the Hint1 plasmid DNA also inhibited AP-1-luciferase reporter activity in WT mouse embryo fibroblast (MEF) studies, and studies with JNK1 deleted or JNK2 deleted MEFs confirmed the essential role for JNK2, but not JNK1, in mediating AP-1 activity. Recent studies indicate that the protein plenty of SH3 (POSH) provides a scaffold that enhances JNK activity. We found that cotransfection of a plasmid DNA encoding POSH stimulated the phosphorylation of c-Jun and also AP-1 reporter activity, and cotransfection with Hint1 inhibited both of these activities. Furthermore, coimmunoprecipitation studies provided evidence that HINT1 forms an in vivo complex with POSH and JNK. These results suggest that HINT1 inhibits AP-1 activity by binding to a POSH-JNK2 complex, thus inhibiting the phosphorylation of c-Jun. This effect could contribute to the tumor suppressor activity of HINT1.

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[18]
Korsisaari N,Makela TP.Interactions of Cdk7 and Kin28 with Hint/PKCI-1 and Hnt1 histidine triad proteins[J].J Biol Chem,2000,275(45):34837-34840. doi:10.1074/jbc.C000505200.Cyclin-dependent kinase 7 (Cdk7) forms a trimeric complex with cyclin H and Mat1 to form the mammalian Cdk-activating kinase, CAK, as well as a part of the basal transcription factor TFIIH, where Cdk7 phosphorylates the C-terminal domain (CTD) of the large subunit of RNA polymerase II. Here, we report a novel interaction between Cdk7 and a histidine triad (HIT) family protein, Hint/PKCI-1. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation and subcellular localization studies, where overexpression of Cdk7 leads to partial relocalization of Hint to the nucleus. The physical association is independent of cyclin H binding or Cdk7 kinase activity and is conserved between the related Sacharomyces cerevisiae CTD kinase Kin28 and the HIT protein Hnt1. Furthermore, combination of a disruption of HNT1 and a KIN28 temperature-sensitive allele in S. cerevisiae led to highly elongated cell morphology and reduced colony formation, indicating a genetic interaction between KIN28 and HNT1. The physical and genetic interactions of Hint and Hnt1 with Cdk7 and Kin28 suggest a role for this class of histidine triad proteins in the regulation of Cdk7 and Kin28 functions.

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[19]
Lee YN,Nechushtan H,Figov N,et al.The function of lysyl-tRNA synthetase and Ap4A as signaling regulators of MITF activity in FcepsilonRI-activated mast cells[J].Immunity,2004,20(2):145-151.Abstract The involvement of microphthalmia transcription factor (MITF) in the function of mast cells, melanocytes, and osteoclasts has recently started to be investigated in depth. In a previous study, we found Hint to be associated with MITF in mast cells and showed that it suppresses MITF's transcriptional activity. Here, we have found that lysyl-tRNA synthetase (LysRS) is also associated with MITF and forms a multicomplex with MITF and Hint. We have also shown that Ap4A, an endogenous molecule consisting of two adenosine linked by four phosphate which is known to be synthesized by LysRS, is accumulated intracellularily above 700 microM in IgE-Ag-activated mast cells, binds to Hint, liberates MITF, and thus leads to the activation of MITF-dependent gene expression. This implies that LysRS plays a key role via Ap4A as an important signaling molecule in MITF transcriptional activity.

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[20]
Razin E,Zhang ZC,Nechushtan H,et al.Suppression of microphthalmia transcriptional activity by its association with protein kinase C-interacting protein 1 in mast cells[J].J Biol Chem,1999,274(48):34272-34276.Abstract Microphthalmia (mi) is a transcription factor that plays a major role in the regulation of growth and function in mast cells and melanocytes. Association of mi with other proteins is a critical step in the regulation of mi-mediated transcriptional activation. We found protein kinase C-interacting protein 1 (PKCI) specifically associated with mi in yeast two-hybrid screening. Immunoprecipitation of mi from quiescent rat basophilic leukemic cells or mouse melanocytes resulted in the specific co-immunoprecipitation of PKCI. This association was significantly reduced on engagement of the surface FcepsilonRI of mast cells or engagement of the Kit receptor on melanocytes. Hence, cell activation caused disengagement of mi from PKCI. Microphthalmia was previously shown to activate the mouse mast cell protease 6 (mMCP-6) promoter. Cotransfection of mi with PKCI in NIH 3T3 fibroblasts containing an mMCP-6 promoter-luciferase reporter demonstrated an up to 94% inhibition of mi-mediated transcriptional activation. PKCI by itself, although localized in the cytosol and nucleus of the cells, has no known physiological function and did not demonstrate transcriptional activity. Its ability to suppres mi transcriptional activity in the transient transfected fibroblast system suggests that it can function in vivo as a negative regulator of mi-induced transcriptional activation.

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[21]
Lee YN,Razin E.Nonconventional involvement of LysRS in the molecular mechanism of USF2 transcriptional activity in FcepsilonRI-activated mast cells[J].Mol Cell Biol,2005,25(20):8904-8912.doi:10.1128/mcb.25.20.8904-8912.2005.Reports of the biological multifunctional activity of various aminoacyl tRNA synthetases have recently accumulated in the literature. The primary function of these critical enzymes is to charge various tRNAs with their appropriate amino acids, thus producing the building blocks of protein synthesis. We have previously shown that lysyl tRNA synthetase (LysRS) associates with microphthalmia transcription factor (MITF) and regulates its activity by synthesis of Ap(4)A in mast cells. Here, we show for the first time that LysRS associates with another transcription factor, USF2, which unlike MITF, is ubiquitously expressed in eukaryotic cells. Using mast cells, we have found that USF2 is negatively regulated by Hint and Ap(4)A acts as a positive regulator of USF2 by a molecular mechanism similar to that described for MITF. Since USF2 plays a significant role in a variety of cellular functions, our finding suggests that LysRS and Ap(4)A may be involved in general regulation of gene transcription.

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[22]
Huber O,Weiske J.Beta-catenin takes a HIT[J].Cell Cycle,2008,7(10):1326-1331.doi:10.4161/cc.7.10.5926.Abstract Histidine triad (HIT) proteins represent a small family of nucleotide-binding and -hydrolyzing proteins, which attracted the attention of cancer biologists because their expression is lost in multiple human malignancies. To some of the family members including Fhit, Hint1 and Hint2, a tumor suppressive activity was assigned. Although highly similar in structure, their mode of action appears to be different as they are not able to compensate each other's function. Surprisingly, in any reported assay system the enzymatic activity of the histidine triad proteins was not required for their tumor suppressor function. Until recently, little was known about the molecular mechanisms mediating the tumor suppressor activities of histidine triad proteins. The identification of new interaction partners started to shed light on the signaling pathways modulated by the HIT proteins. Here, we summarize these findings with special emphasis on the histidine triad proteins Hint1 and Fhit and their repressive activity on the beta-catenin signaling function.

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[23]
Wu XS,Bao TH,Ke Y,et al.Hint1 suppresses migration and invasion of hepatocellular carcinoma cells in vitro by modulating girdin activity[J].Tumour Biol,2016,37(11):14711-14719.doi:10.1007/s13277-016-5336-z.Histidine triad nucleotide-binding protein 1 (Hint1) is a haploinsufficient tumor suppressor gene. Its role in cancer cell migration has not been previously speculated. In the current study, we examined the expression of Hint1 in metastatic and non-metastatic lymph nodes of hepatocellular carcinoma (HCC) patients and further elucidated the effect of Hint1 expression on girdin expression and phosphorylation of AKT and ERK1/2 and on the migration of HCC cells in vitro. Expression of Hint1 and girdin in primary HCC tissues and metastatic and non-metastatic lymph nodes was determined by RT-PCR assays. HepG2 cells were transfected with plasmid vectors overexpressing Hint1 or small interfering RNA (siRNA) targeting Hint1, girdin, Hint1 plus girdin, or the scrambled RNA. Migration and invasion of HCC cells were examined by wound and Transwell assays. Protein expression was detected by immunofluorescence and immunoblotting assays. RT-PCR assays revealed that the messenger RNA (mRNA) transcript levels of Hint1 were markedly lower than those of primary HCC tissues and non-metastatic lymph nodes (P < 0.01). By contrast, the mRNA transcript levels of girdin were significantly higher than non-metastatic lymph nodes (P < 0.05). Furthermore, siRNA knockdown of HINT1 resulted in a significant increase in the mRNA transcript levels of girdin in HepG2 cells (P < 0.05). Wound assays and Transwell assays showed that Hint1 knockdown by siRNA significantly enhanced the migration and invasion of HepG2 cells compared to HepG2 cells transfected with scrambled siRNA. Hint1 knockdown also led to significantly increased phosphorylation of girdin and AKT in HepG2 cells (P < 0.05), which, however, was effectively aborted by girdin knockdown by siRNA (P < 0.05). Hint1 is downregulated in metastatic lymph nodes and is implicated in migration and invasion of HCC cells in vitro by modulating girdin and AKT expression and phosphorylation. The Hint1-girdin-AKT signaling axis should be further dissected for its role in HCC migration and invasion and may be therapeutically targeted to suppress tumor growth and metastasis.

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[24]
Shi Z,Wu X,Ke Y,et al.Hint1 up-regulates IkappaBalpha by targeting the beta-TrCP subunit of SCF E3 ligase in human hepatocellular carcinoma cells[J].Dig Dis Sci,2016,61(3):785-794.doi:10.1007/s10620-015-3927-y.BACKGROUND AND AIM: There is increasing evidence that histidine triad nucleotide-binding protein 1 (HINT1) is a novel tumor suppressor. In the present study, we investigated the mechanism by which HINT1 promotes the stability of inhibitor of NF-kappaB alpha (IkappaBalpha) in the cytoplasm of hepatocellular carcinoma (HCC) cells, which was observed in our previous study (Wang et al. in Int J Cancer 124:1526-1534, 2009). METHODS: We examined HINT1 and IkappaBalpha expression in HCC cell lines and determined the effect of HINT1 overexpression and knockdown on IkappaBalpha protein and mRNA expression in these cell lines. Then, ubiquitination assays were performed to investigate the effects of HINT1 expression plasmid transfection on IkappaBalpha ubiquitination. Next, the interaction between HINT1 and beta-TrCP was investigated in immunoprecipitation and immunofluorescence assays. RESULTS: Our data showed that increased HINT1 expression in HepG2 and SMMC7702 cells markedly increased IkappaBalpha protein levels, while decreased HINT1 expression markedly decreased them. Overexpression or knockdown of HINT1 did not alter the transcription of IkappaBalpha, but HINT1 inhibited proteasomal IkappaBalpha degradation and reduced its ubiquitination levels. This inhibition might occur because HINT1 is a component of the SCFbeta-TrCP E3 ligase, which is responsible for IkappaBalpha ubiquitination and degradation. CONCLUSION: This study provides new evidence that HINT1 is a regulator of IkappaBalpha through SCFbeta-TrCP E3 ligase. These findings help to clarify the mechanism underlying the anticancer effects of HINT1.

[25]
Weiske J,Huber O.The histidine triad protein Hint1 triggers apoptosis independent of its enzymatic activity[J].J Biol Chem,2006,281(37):27356-27366.doi:10.1074/jbc.M513452200.Abstract Hint1 is a member of the evolutionarily conserved family of histidine triad proteins that acts as a haplo-insufficient tumor suppressor inducing spontaneous tumor formation in Hint+/- and Hint-/- mouse models. However, the molecular mechanisms for the tumor-suppressing activity are poorly defined. In this respect, we have recently shown that Hint1, by interaction with Pontin and Reptin, inhibits T-cell factor/beta-catenin-mediated transcription of Wnt target genes. In this study, we have found that, after transient transfection with Hint1, SW480 and MCF-7 cells undergo apoptosis as analyzed by pro-caspase-3 and poly(ADP-ribose) polymerase cleavage, M30 CytoDEATH staining, cytochrome c release, and DNA fragmentation enzyme-linked immunosorbent assay. Hint1 is involved in the regulation of apoptotic pathways by inducing an up-regulation of p53 expression coinciding with an up-regulation of the proapoptotic factor Bax and a concomitant down-regulation of the apoptosis inhibitor Bcl-2. Bad and Puma levels remained unchanged. Further analyses revealed that Hint1 is associated with the Bax promoter and is a component of the Tip60 histone acetyltransferase complex and, in this context, appears to be involved in the regulation of Bax expression. Knockdown of Hint1 by short hairpin RNA resulted in down-regulation of p53 and Bax but had no effect on Bcl-2 expression. A mutant Hint1 (H112N) protein defective in enzymatic activity as an AMP-NH2 hydrolase was not impaired in induction of apoptosis, suggesting that the Hint1 pro-apoptotic activity is independent of the Hint1 enzymatic activity.

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[26]
Linde CI,Feng B,Wang JB,et al.Histidine triad nucleotide-binding protein 1(HINT1) regulates Ca(2+) signaling in mouse fibroblasts and neuronal cells via store-operated Ca(2+) entry pathway[J].Am J Physiol Cell Physiol,2013,304(11):C1098-1104.doi:10.1152/ajpcell.00073.2013.

[27]
Scholer J,Ferralli J,Thiry S,et al.The intracellular domain of teneurin-1 induces the activity of microphthalmia-associated transcription factor(MITF) by binding to transcriptional repressor HINT1[J].J Biol Chem,2015,290(13):8154-8165.doi:10.1074/jbc.M114.615922.Teneurins are large type II transmembrane proteins that are necessary for the normal development of the CNS. Although many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus, where it can influence gene transcription. Because teneurin ICDs do not contain any intrinsic DNA binding sequences, interaction partners are required to affect transcription. Here, we identified histidine triad nucleotide binding protein 1 (HINT1) as a human teneurin-1 ICD interaction partner in a yeast two-hybrid screen. This interaction was confirmed in human cells, where HINT1 is known to inhibit the transcription of target genes by directly binding to transcription factors at the promoter. In a whole transcriptome analysis of BS149 glioblastoma cells overexpressing the teneurin-1 ICD, several microphthalmia-associated transcription factor (MITF) target genes were found to be up-regulated. Directly comparing the transcriptomes of MITF versus TEN1-ICD-overexpressing BS149 cells revealed 42 co-regulated genes, including glycoprotein non-metastatic b (GPNMB). Using real-time quantitative PCR to detect endogenous GPNMB expression upon overexpression of MITF and HINT1 as well as promoter reporter assays using GPNMB promoter constructs, we could demonstrate that the teneurin-1 ICD binds HINT1, thus switching on MITF-dependent transcription of GPNMB.

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[28]
Sanchez-Blazquez P,Rodriguez-Munoz M,Bailon C,et al.GPCRs promote the release of zinc ions mediated by nNOS/NO and the redox transducer RGSZ2 protein[J].Antioxid Redox Signal,2012,17(9):1163-1177.doi:10.1089/ars.2012.4517.Morphine signaling via the μ-opioid receptor (MOR) is coupled to redox-dependent zinc release from endogenous stores. Thus, MOR activation stimulates the complex formed by RGSZ2 (a regulator of G protein signaling) and neural nitric oxide synthase (nNOS) to produce NO, and to recruit PKCγ and Raf-1 in a zinc-dependent manner. Accordingly, we investigated whether redox regulation of zinc metabolism was unique to the MOR, or if it is a signaling mechanism shared by G-protein coupled receptors (GPCRs).A physical interaction with the RGSZ2-nNOS complex was detected for the following GPCRs: neuropeptides, MOR and δ-opioid (DOR); biogenic amines, 5HT1A, 5HT2A, α2A, D1 and D2; acetylcholine, muscarinic M2 and M4; excitatory amino acid glutamate, mGlu2 and mGlu5; and derivatives of arachidonic acid (anandamide), CB1. Agonist activation of these receptors induced the release of zinc ions from the RGSZ2 zinc finger via a nNOS/NO-dependent mechanism, recruiting PKCγ and Raf-1 to the C terminus or the third internal loop of the GPCR. Innovation: A series of GPCRs share an unexpected mechanistic feature, the nNOS/NO-dependent regulation of zinc ion signaling via a redox mechanism. The RGSZ2 protein emerges as a potential redox zinc switch that converts NO signals into zinc signals, thereby able to modulate the function of redox sensor proteins like PKCγ or Raf-1.Redox mechanisms are crucial for the successful propagation of GPCR signals in neurons. Thus, dysfunctions of GPCR-regulated NO/zinc signaling may contribute to neurodegenerative and mood disorders such as Alzheimer's disease and depression.

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[29]
Rodriguez-Munoz M,de la Torre-Madrid E,Sanchez-Blazquez P,et al. NO-released zinc supports the simultaneous binding of Raf-1 and PKCgamma cysteine-rich domains to HINT1 protein at the mu-opioid receptor[J].Antioxid Redox Signal,2011,14(12):2413-2425.doi:10.1089/ars.2010.3511.In the brain, the mu-opioid receptor (MOR) activates neural nitric oxide synthase (nNOS) through the PI3K/Akt pathway. The resulting nitric oxide (NO) enhances the function of the glutamate N-methyl-d-aspartate receptor (NMDAR)/calcium and calmodulin-dependent serine/threonine kinase (CaMKII), which subsequently diminishes MOR signaling strength. Because the ERK1/2 cascade is implicated in opioid tolerance, we analyzed the role of morphine-generated NO in this negative regulation. We found that NO-released endogenous zinc ions recruit the Ras/Raf-1/ERK1/2 cassette to histidine triad nucleotide-binding protein 1 (HINT1). A-Raf and B-Raf showed little or no MOR association. The zinc ions bridge the Raf-1 cysteine-rich domain (CRD) with HINT1 at the MOR C-terminus. Morphine also recruits PKC纬 via NO/zinc to the MOR-HINT1 complex. Both Raf-1 and PKC纬 CRDs bind simultaneously to HINT1, enabling PKC纬 to enhance Raf-1 function to intensify MEK/ERK1/2 activation. Thus, through attached HINT1, the MOR facilitates the cross-talk of two NO- and zinc-regulated signal-transduction pathways, PKC/Src and Raf-1/ERK1/2, implicated in the negative control of morphine effects. This study reveals new aspects of ERK1/2 regulation by the MOR without requiring the transactivation of a receptor tyrosine kinase.

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[30]
Rodriguez-Munoz M,de la Torre-Madrid E,Sanchez-Blazquez P,et al. NMDAR-nNOS generated zinc recruits PKCgamma to the HINT1-RGS17 complex bound to the C terminus of Mu-opioid receptors[J].Cell Signal,2008,20(10):1855-1864.doi:10.1016/j.cellsig.2008.06.015.In neurons, the C terminus of the Mu-opioid receptor (MOR) binds to the protein kinase C-interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) which in turn binds the regulator of G-protein signalling RGSZ1/Z2 (RGSZ) protein. In this study, we found that intracerebroventricular (icv) administration of morphine recruits PKC isoforms, mostly PKC gamma, to the MOR via the HINT1/RGSZ complex There, diacylglycerol (DAG) activates this PKC gamma to phosphorylate the MOR and thus, its signal strength was reduced. When PKCI/HINT1 expression is depressed, morphine produces stronger analgesic effects and neither the PKC gamma-MOR complex nor serine phosphorylation of this receptor is detected. This MOR-PKC association involves the cysteine rich domains (CRDs) in the regulatory C1 region of PKC, as well as requiring free zinc ions, HINT1 and RGSZ proteins. Increasing the availability of this metal ion recruits inactive PKC gamma to the MOR, while phorbol esters prevent this binding and even disrupt it The nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) foments the association of PKC gamma with the MORs, effect that was prevented by the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN). suggesting a role for endogenous zinc and neural nitric oxide synthase. The N-methyl-D-aspartate receptor (NMDAR) antagonist, MK801, also prevented PKC gamma recruitment to MORs and serine phosphorylation of the receptors following icv morphine. These results indicate that the NMDAR/nNOS cascade, activated via MORs, provide the free zinc ions required for inactive PKC gamma to bind to HINT1/RGSZ complex at the C terminus of the receptor. (C) 2008 Elsevier Inc. All rights reserved.

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[31]
Ajit SK,Ramineni S,Edris W,et al.RGSZ1 interacts with protein kinase C interacting protein PKCI-1 and modulates mu opioid receptor signaling[J].Cell Signal,2007,19(4):723-730.doi:10.1016/j.cellsig.2006.09.008.Protein kinase C interacting protein (PKCI-1) was identified among the potential interactors from a yeast two hybrid screen of human brain library using N terminal of RGSZ1 as a bait. The cysteine string region, unique to the RZ subfamily, contributes to the observed interaction because PKCI-1 interacted with N-terminus of RGS17 and GAIP, but not with that of RGS2 or RGS7 where cysteine string motif is absent. The interaction between RGSZ1 and PKCI-1 was confirmed by coinummoprecipitation and immuno fluorescence. PKCI-1 and RGSZ1 could be detected by coimmunoprecipitation using 14-3-3 antibody in cells transfected with PKCI-1 or RGSZ1 respectively, but when transfected with PKCI-1 and RGSZ1 together, only RGSZ1 could be detected. Phosphorylation of G alpha z by protein kinase C (PKC) reduces the ability of the RGS to effectively function as GTPase accelerating protein for G alpha z, and interferes with ability of Getz to interact with beta gamma complex. We investigated the roles of 14-3-3 and PKCI-1 in phosphorylation of G alpha z. Phosphorylation of G alpha z by PKC was inhibited by 14-3-3 and the presence of PKCI-1 did not provide any further inhibition. PKCI-1 interacts with mu opioid receptor and suppresses receptor desensitization and PKC related mu opioid receptor phosphorylation [W. Guang, H. Wang, T. Su, I.B. Weinstein, J.B. Wang, Mol. Pharmacol. 66 (2004) 1285.]. Previous studies have also shown that mu opioid receptor co-precipitates with RGSZ1 and influence mu receptor signaling by acting as effector antagonists [J. Garzon, M. Rodriguez-Munoz, P. Sanchez-Blazquez, Neurophannacology 48 (2005) 853., J. Garzon, M. Rodriguez-Munoz, A. Lopez-Fando, P. Sanchez-Blazquez Neuropsychopharmacology 30 (2005) 1632.]. Inhibition of cAMP by mu opioid receptor was significantly reduced by RGSZ1 and this effect was enhanced in combination with PKCI-1. Our studies thus provide a link between the previous observations mentioned above and indicate that the major function of

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[32]
Guang W,Wang H,Su T,et al.Role of mPKCI,a novel mu-opioid receptor interactive protein,in receptor desensitization,phosphorylation,and morphine-induced analgesia[J].Mol Pharmacol,2004,66(5):1285-1292.doi:10.1124/mol.66.5.

[33]
Rodriguez-Munoz M,Sanchez-Blazquez P,Vicente-Sanchez A,et al.The histidine triad nucleotide-binding protein 1 supports mu-opioid receptor-glutamate NMDA receptor cross-regulation[J].Cell Mol Life Sci,2011,68(17):2933-2949.doi:10.1007/s00018-010-0598-x.A series of pharmacological and physiological studies have demonstrated the functional cross-regulation between MOR and NMDAR. These receptors coexist at postsynaptic sites in midbrain periaqueductal grey (PAG) neurons, an area implicated in the analgesic effects of opioids like morphine. In this study, we found that the MOR-associated histidine triad nucleotide-binding protein 1 (HINT1) is essential for maintaining the connection between the NMDAR and MOR. Morphine-induced analgesic tolerance is prevented and even rescued by inhibiting PKC or by antagonizing NMDAR. However, in the absence of HINT1, the MOR becomes supersensitive to morphine before suffering a profound and lasting desensitization that is refractory to PKC inhibition or NMDAR antagonism. Thus, HINT1 emerges as a key protein that is critical for sustaining NMDAR-mediated regulation of MOR signaling strength. Thus, HINT1 deficiency may contribute to opioid-intractable pain syndromes by causing long-term MOR desensitization via mechanisms independent of NMDAR.

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[34]
Rodriguez-Munoz M,Garzon J.Nitric oxide and zinc-mediated protein assemblies involved in mu opioid receptor signaling[J].Mol Neurobiol,2013,48(3):769-782.doi:10.1007/s12035-013-8465-z.Opioids are among the most effective analgesics in controlling the perception of intense pain, although their continuous use decreases their potency due to the development of tolerance. The glutamate N-methyl-D-aspartate (NMDA) receptor system is currently considered to be the most relevant functional antagonist of morphine analgesia. In the postsynapse of different brain regions the C terminus of the mu-opioid receptor (MOR) associates with NR1 subunits of NMDARs, as well as with a series of signaling proteins, such as neural nitric oxide synthase (nNOS)/nitric oxide (NO), protein kinase C (PKC), calcium and calmodulin-dependent kinase II (CaMKII) and the mitogen-activated protein kinases (MAPKs). NO is implicated in redox signaling and PKC falls under the regulation of zinc metabolism, suggesting that these signaling elements might participate in the regulation of MOR activity by the NMDAR. In this review, we discuss the influence of redox signaling in the mechanisms whose plasticity triggers opioid tolerance. Thus, the MOR C terminus assembles a series of signaling proteins around the homodimeric histidine triad nucleotide-binding protein 1 (HINT1). The NMDAR NR1 subunit and the regulator of G protein signaling RGSZ2 bind HINT1 in a zinc-independent manner, with RGSZ2 associating with nNOS and regulating MOR-induced production of NO. This NO acts on the RGSZ2 zinc finger, providing the zinc ions that are required for PKC/Raf-1 cysteine-rich domains to simultaneously bind to the histidines present in the HINT1 homodimer. The MOR-induced activation of phospholipase 尾 (PLC尾) regulates PKC, which increases the reactive oxygen species (ROS) by acting on NOX/NADPH, consolidating the long-term PKC activation required to regulate the Raf-1/MAPK cascade and enhancing NMDAR function. Thus, RGSZ2 serves as a Redox Zinc Switch that converts NO signals into Zinc signals, thereby modulating Redox Sensor Proteins like PKC纬 and Raf-1. Accordingly, redox-dependent and independent processes weave together to situate the MOR under the negative control of the NMDAR.

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[35]
Garzon J,Rodriguez-Munoz M,Sanchez-Blazquez P.Direct association of Mu-opioid and NMDA glutamate receptors supports their cross-regulation:molecular implications for opioid tolerance[J].Curr Drug Abuse Rev,2012,5(3):199-226.

[36]
Rodriguez-Munoz M,Cortes-Montero E,Pozo-Rodrigalvarez A,et al.The ON:OFF switch,sigma1R-HINT1 protein,controls GPCR-NMDA receptor cross-regulation:implications in neurological disorders[J].Oncotarget,2015,6(34):35458-35477.doi:10.18632/oncotarget.6064.In the brain, the histidine triad nucleotide-binding protein 1 (HINT1) and sigma 1 receptors (σ1Rs) coordinate the activity of certain G-protein coupled receptors (GPCRs) with that of glutamate N-methyl-D-aspartate receptors (NMDARs). To determine the role of HINT1-σ1R in the plasticity of GPCR-NMDAR interactions, substances acting at MOR, cannabinoid CB1 receptor, NMDAR and σ1R were injected into mice, and their effects were evaluated through in vivo, ex vivo, and in vitro assays. It was observed that HINT1 protein binds to GPCRs and NMDAR NR1 subunits in a calcium-independent manner, whereas σ1R binding to these proteins increases in the presence of calcium. In this scenario, σ1R agonists keep HINT1 at the GPCR and stimulate GPCR-NMDAR interaction, whereas σ1R antagonists transfer HINT1 to NR1 subunits and disengage both receptors. This regulation is lost in σ1R-/- mice, where HINT1 proteins mostly associate with NMDARs, and GPCRs are physically and functionally disconnected from NMDARs. In HINT1-/- mice, ischemia produces low NMDAR-mediated brain damage, suggesting that several different GPCRs enhance glutamate excitotoxicity via HINT1-σ1R. Thus, several GPCRs associate with NMDARs by a dynamic process under the physiological control of HINT1 proteins and σ1Rs. The NMDAR-HINT1-σ1R complex deserves attention because it offers new therapeutic opportunities.

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[37]
Dang YH,Liu P,Ma R,et al.HINT1 is involved in the behavioral abnormalities induced by social isolation rearing[J].Neurosci Lett,2015,607:40-45.doi:10.1016/j.neulet.2015.08.026.Social isolation (SI) rearing has been demonstrated to induce behavioral abnormalities like anxiety, impulsivity, aggression, and learning and memory deficits which are relevant to core symptoms in patients with some certain neuropsychiatric disorders. But the underlying pathophysiological mechanisms remain unclear. Recent studies have revealed HINT1 has close relation with diverse neuropsychiatric diseases. In this present study, the SI rearing mice exhibited depression-like and aggressive behavior. Besides, HINT1 protein levels decreased in PFC but increased in HIP. Based on the data obtained, we concluded that HINT1 is involved in the behavioral abnormalities induced by social isolation and exerts distinct roles in different encephalic regions.

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[38]
Baron M.Genetics of schizophrenia and the new millennium:progress and pitfalls[J].Am J Hum Genet,2001,68(2):299-312.doi:10.1086/318212.Artificial selection for intrinsic aerobic endurance running capacity was started using genetically heterogeneous N:NIH stock of rats as a founder population (n = 168). Selection for low and high capacity was based upon distance run to exhaustion on a motorized treadmill using a velocity-ramped running protocol. The starting velocity was 10 m/min and was increased by 1 m/min every 2 min (slope was constant at 15 degrees). At each generation, within-family selection was practiced using 13 families for both the low and high lines. A rotational breeding paradigm maintained the coefficient of inbreeding at less than 1% per generation. On average the founder population ran to exhaustion in 355 +/- 11 m. Six generations of selection produced lines that differed in running capacity by 171%, with most of the change occurring in the high line. At generation 6 the low line ran 310 +/- 8 m and the high line 839 +/- 21 m at exhaustion. Selection for running capacity produced changes in body weight as a correlated trait. By generation 6, the low-line females were 20% heavier than the high-line females, and the low-line males were 16% heavier than the high-line males.

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[39]
Straub RE,MacLean CJ,O’Neill FA,et al. Support for a possible schizophrenia vulnerability locus in region 5q22-31 in Irish families[J].Mol Psychiatry,1997,2(2):148-155.In our genome scan for genes in 265 Irish pedigrees, marker D5S818 in 5q22 produced the second best result of the first 223 markers tested (P = 0.002). We then tested an additional 13 markers and the evidence suggests the presence of a vulnerability locus for in region 5q22-31. This region appears to be distinct from those 5 regions studied in two prior reports, but the same as that producing positive results in the report by Wildenauer and colleagues found elsewhere in this issue. The largest pairwise heterogeneity LOD (H-LOD) score was found with marker D5S393 (max 3.04, P = 0.0005), assuming a narrow phenotypic category, and a genetic model with intermediate heterozygotic liability. In marked contrast to the H-LOD scores from our sample with markers from the regions of interest on 6p and 8p, expanding the disease definition to include spectrum or nonspectrum disorders produced substantially smaller scores, with a number of markers failing to yield positive values at any recombination fraction. Using multipoint H-LODS, the strongest evidence for linkage occurs under the narrow phenotypic definition and recessive genetic model, with a peak at marker D5S804 (max 3.35, P = 0.0002). Multipoint nonparametric linkage analysis produced a peak in the same location (max z = 2.84, P = 0.002) with the narrow phenotypic definition. This putative vulnerability locus appears to be segregating in 10-25% of the families studied, but this estimate is tentative. Comparison of individual family multipoint H-LOD scores at the regions of interest on 6p, 8p and 5q showed that only a minority of families yield high lod scores in two or three regions.

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[40]
Nestler EJ,Pena CJ,Kundakovic M,et al.Epigenetic basis of mental illness[J].Neuroscientist,2016,22(5):447-463.doi:10.1177/1073858415608147.Psychiatric disorders are complex multifactorial illnesses involving chronic alterations in neural circuit structure and function as well as likely abnormalities in glial cells. While genetic factors are important in the etiology of most mental disorders, the relatively high rates of discordance among identical twins, particularly for depression and other stress-related syndromes, clearly indicate the importance of additional mechanisms. Environmental factors such as stress are known to play a role in the onset of these illnesses. Exposure to such environmental insults induces stable changes in gene expression, neural circuit function, and ultimately behavior, and these maladaptations appear distinct between developmental versus adult exposures. Increasing evidence indicates that these sustained abnormalities are maintained by epigenetic modifications in specific brain regions. Indeed, transcriptional dysregulation and the aberrant epigenetic regulation that underlies this dysregulation is a unifying theme in psychiatric disorders. Here, we provide a progress report of epigenetic studies of the three major psychiatric syndromes, depression, schizophrenia, and bipolar disorder. We review the literature derived from animal models of these disorders as well as from studies of postmortem brain tissue from human patients. While epigenetic studies of mental illness remain at early stages, understanding how environmental factors recruit the epigenetic machinery within specific brain regions to cause lasting changes in disease susceptibility and pathophysiology is revealing new insight into the etiology and treatment of these conditions.

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[41]
Vawter MP,Crook JM,Hyde TM,et al.Microarray analysis of gene expression in the prefrontal cortex in schizophrenia:a preliminary study[J].Schizophr Res,2002,58(1):11-20.

[42]
Vawter MP,Barrett T,Cheadle C,et al.Application of cDNA microarrays to examine gene expression differences in schizophrenia[J].Brain Res Bull,2001,55(5):641-650.Using cDNA microarrays we have investigated gene expression patterns in brain regions of patients with schizophrenia. A cDNA neuroarray, comprised of genes related to brain function, was used to screen pools of samples from the cerebellum and prefrontal cortex from a matched set of subjects, and middle temporal gyrus, from a separate subject cohort. Samples of cerebellum and prefrontal cortex from neuroleptic naive patients were also included. Genes that passed a 3% reproducibility criterion for differential expression in independent experiments included 21 genes for drug-treated patients and 5 genes for drug-naive patients. Of these 26 genes, 10 genes were increased and 16 were decreased. Many of the differentially expressed genes were related to synaptic signaling and proteolytic functions. A smaller number of these genes were also differentially expressed in the middle temporal gyrus. The five genes that were differentially expressed in two brain regions from separate cohorts are: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; sialyltransferase; proteasome subunit, alpha type 1; ubiquitin carboxyl-terminal esterase L1; and solute carrier family 10, member 1. Identification of patterns of changes in gene expression may lead to a better understanding of the pathophysiology of schizophrenia disorders.

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[43]
Vawter MP,Shannon Weickert C,Ferran E,et al.Gene expression of metabolic enzymes and a protease inhibitor in the prefrontal cortex are decreased in schizophrenia[J].Neurochem Res,2004,29(6):1245-1255.

[44]
Chen X,Wang X,Hossain S,et al.Haplotypes spanning SPEC2,PDZ-GEF2 and ACSL6 genes are associated with schizophrenia[J].Hum Mol Genet,2006,15(22):3329-3342.doi:10.1093/hmg/ddl409.Chromosome 5q22-33 is a region where studies have repeatedly found evidence for linkage to schizophrenia. In this report, we took a stepwise approach to systematically map this region in the Irish Study of High Density Schizophrenia Families (ISHDSF, 267 families, 1337 subjects) sample. We typed 289 SNPs in the critical interval of 8 million basepairs and found a 758 kb interval coding for the SPEC2/PDZ-GEF2/ACSL6 genes to be associated with the disease. Using sex and genotype-conditioned transmission disequilibrium test analyses, we found that 19 of the 24 typed markers were associated with the disease and the associations were sex-specific. We replicated these findings with an Irish case-control sample (657 cases and 414 controls), an Irish parent-proband trio sample (187 families, 564 subjects), a German nuclear family sample (211 families, 751 subjects) and a Pittsburgh nuclear family sample (247 families, 729 subjects). In all four samples, we replicated the sex-specific associations at the levels of both individual markers and haplotypes using sex- and genotype-conditioned analyses. Three risk haplotypes were identified in the five samples, and each haplotype was found in at least two samples. Consistent with the discovery of multiple estrogen-response elements in this region, our data showed that the impact of these haplotypes on risk for schizophrenia differed in males and females. From these data, we concluded that haplotypes underlying the SPEC2/PDZ-GEF2/ACSL6 region are associated with schizophrenia. However, due to the extended high LD in this region, we were unable to distinguish whether the association signals came from one or more of these genes.

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[45]
Varadarajulu J,Schmitt A,Falkai P,et al.Differential expression of HINT1 in schizophrenia brain tissue[J].Eur Arch Psychiatry Clin Neurosci,2012,262(2):167-172.doi:10.1007/s00406-011-0216-4.

[46]
Kurotaki N,Tasaki S,Mishima H,et al.Identification of novel schizophrenia loci by homozygosity mapping using DNA microarray analysis[J].PLoS One,2011,6(5):e20589.doi:10.1371/journal.pone.0020589.

[47]
Barbier E,Zapata A,Oh E,et al.Supersensitivity to amphetamine in protein kinase-C interacting protein/HINT1 knockout mice[J].Neuropsychopharmacology,2007,32(8):1774-1782.doi:10.1038/sj.npp.1301301.Protein kinase C interacting protein/histidine triad nucleotide binding protein I (PKCI/HINTI) is a member of the histidine triad protein family. Although this protein is widely expressed in the mammalian brain including mesocorticolimbic and mesostriatal regions, its physiological function in CNS remains unknown. Recent microarray studies reported decreased mRNA expression of PKCI/HINT in the frontal cortex of individuals with schizophrenia, suggesting the possible involvement of this protein in the pathophysiology of the disease. In view of the documented link between dopamine (DA) transmission and schizophrenia, the present study used behavioral and neurochemical approaches to examine the influence of constitutive PKCI/HINTI deletion upon: (i) basal and amphetamine (AMPH)-evoked locomotor activity; (ii) DA dynamics in the dorsal striatum, and (iii) postsynaptic DA receptor function. PKCI/HINTI

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[48]
Ross CA,Margolis RL,Reading SA,et al.Neurobiology of schizophrenia[J].Neuron,2006,52(1):139-153.doi:10.1016/j.neuron.2006.09.015.

[49]
van den Buuse M,Garner B,Gogos A,et al. Importance of animal models in schizophrenia research[J].Aust N Z J Psychiatry,2005,39(7):550-557.doi:10.1111/j.1440-1614.2005.01626.x.This review aims to summarize the importance of animal models for research on psychiatric illnesses, particularly schizophrenia.Several aspects of animal models are addressed, including animal experimentation ethics and theoretical considerations of different aspects of validity of animal models. A more specific discussion is included on two of the most widely used behavioural models, psychotropic drug-induced locomotor hyperactivity and prepulse inhibition, followed by comments on the difficulty of modelling negative symptoms of schizophrenia. Furthermore, we emphasize the impact of new developments in molecular biology and the generation of genetically modified mice, which have generated the concept of behavioural phenotyping.Complex psychiatric illnesses, such as schizophrenia, cannot be exactly reproduced in species such as rats and mice. Nevertheless, by providing new information on the role of neurotransmitter systems and genes in behavioural function, animal 'models' can be an important tool in unravelling mechanisms involved in the symptoms and development of such illnesses, alongside approaches such as post-mortem studies, cognitive and psychophysiological studies, imaging and epidemiology.

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[50]
Skre H.Genetic and clinical aspects of Charcot-Marie-Tooth’s disease[J].Clin Genet,1974,6(2):98-118.The prevalence of Charcot-Marie-Tooth's disease (CMT) was studied in Western Norway, an area with several isolated districts with a population of 725,000 (1968). Three hereditary types were distinguished in the area: autosomal dominant CMT with an estimated prevalence of 36/100,000; X-linked recessive CMT with a prevalence of 3.6/100,000; and autosomal recessive CMT with a prevalence of 1.4/100,00. Gene frequencies were 3 - 7. 10 -4 , 1 - 9. 10 -4 , and 4 - 8. 10 -4 in autosomal dominant, X-linked, and autosomal recessive CMT, respectively, while the corresponding mutation rates were 13 - 0, 5 - 5, and 3 - 5 per million gametes per generation. The penetrance was almost complete for all three variants of CMT. Strict diagnostic criteria were followed in the selection of the 37 index cases. A family investigation was carried out with 238 subjects, during which 69 secondary cases were detected. Another 57 subjects had unspecific neuropathy (Un), which did not fit a diagnosis of CMT or other neurological disease. In the diagnosis of Un, a score system was used, with age and sex corrections based on findings in a normal population. Generally, the most severe disease course was found in the recessive CMT types, but there was also more clinical variation, suggesting CNS involvement in some cases (upper motor neuron affection, cerebellar signs). Scoliosis and spinal ataxia were not infrequent, even in cases with autosomal dominant CMT. The prevalence cf Un was highest in the relatives of recessive CMT cases, with a ratio of affected to normal in sibs compatible with a hypothesis of several cases of heterozygous manifestation. In the relatives of autosomal dominant CMT cases, Un prevalence was also higher than in the population, but lower in 2nd degree relatives than in 1st degree; the ratios fitted a hypothesis of polygenic Un inheritance. Significant differences were found in the score patterns of Un in the recessive CMT families and in the autosomal dominant families, suggesting their difference of origin. The reason for clustering of Un cases in autosomal dominant CMT families is obscure, since it can be only partly attributed to early manifestation of CMT. It is suggested that Un and CMT, mainly in autosomal dominant CMT, interact to form a spectrum of differing phenotypes, so explaining the problem of "transitional forms" between CMT and other hereditary nervous disorders. Recessive CMT, being a more generalized nervous disease, attains, through differing expressivity, phenotypes which vary between individual cases.

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[51]
Vance JM.Hereditary motor and sensory neuropathies[J].J Med Genet,1991,28(1):1-5.Abstract Transcranial magnetic brain stimulation was used to study central motor conduction (CMCT) to small hand muscles in patients with peroneal muscular atrophy and hereditary spastic paraplegia at the National Hospital and Institute of Neurology, Queen Square and the Department of Neurological Science, Royal Free Hospital, London, UK.

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[52]
Tazir M,Bellatache M,Nouioua S,et al.Autosomal recessive Charcot-Marie-Tooth disease:from genes to phenotypes[J].J Peripher Nerv Syst,2013,18(2):113-129.doi:10.1111/jns5.12026.The prevalence of Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy (HMSN) varies in different populations. While in some countries of Western Europe, the United States and Japan the dominant form of HMSN is the most frequent, in other countries such as those of the Mediterranean Basin, the autosomal recessive form (AR-CMT) is more common. Autosomal recessive CMT cases are generally characterized by earlier onset, usually before the age of 2 or 3 years, and rapid clinical progression that results in severe polyneuropathy and more marked distal limb deformities such as pes equino-varus, claw-like hands, and often major spinal deformities. Recent clinical, morphological and molecular investigations of CMT families with autosomal recessive inheritance allowed the identification of many genes such as GDAP1, MTMR2, SBF2, NDRG1, EGR2, SH3TC2, PRX, FGD4, and FIG4, implicated in demyelinating forms (ARCMT1 or CMT4), and LMNA, MED25, HINT1, GDAP1, LRSAM1, NEFL, HSPB1 and MFN2 in axonal forms (ARCMT2). However, many patients remain without genetic diagnosis to date, prompting investigations into ARCMT families in order to help discover new genes and common pathways. This review summarizes recent advances regarding the genotypes and corresponding phenotypes of AR-CMT.

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[53]
Zhao H,Race V,Matthijs G,et al.Exome sequencing reveals HINT1 mutations as a cause of distal hereditary motor neuropathy[J].Eur J Hum Genet,2014,22(6):847-850.doi:10.1038/ejhg.2013.231.Abstract Distal hereditary motor neuropathies (dHMNs) are a heterogenous group of genetic disorders with length-dependent degeneration of motor axons. Obtaining a genetic diagnosis in patients with dHMN remains challenging. We performed exome sequencing in a diagnostic setting in 12 patients with a clinical diagnosis of dHMN. Potential disease-causing variants in genes associated with dHMN and other forms of inherited neuropathies/motor neuron diseases were validated using Sequenom. The coverage in the genes studied was >95% with an average coverage of >50 times. In none of the patients a mutations was found in genes previously reported to be associated with dHMN. However, in 2/12 patients a recessive mutation in histidine triad nucleotide binding protein 1 (HINT1, recently discovered as a cause of axonal neuropathy with neuromyotonia) was identified. Our results demonstrate the diagnostic value of exome sequencing for patients with inherited neuropathies. The phenotypic spectrum of recessive mutations in HINT1 includes dHMN. HINT1 should be added to the list of genes to check for in dHMN.

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[54]
Baets J,De Jonghe P,and Timmerman V.Recent advances in Charcot-Marie-Tooth disease[J].Curr Opin Neurol,2014,27(5):532-540.doi:10.1097/WCO.0000000000000131.This article focuses on recent advances in Charcot-Marie-Tooth disease, in particular additions to the genetic spectrum, novel paradigms in molecular techniques and an update on therapeutic strategies.Several new Charcot-Marie-Tooth disease-causing genes have been recently identified, further enlarging the genetic diversity and phenotypic variability, including: SBF1, DHTKD1, TFG, MARS, HARS, HINT1, TRIM1, AIFM1, PDK3 and GNB4. The increasing availability and affordability of next-generation sequencing technologies has ramped up gene discovery and drastically changed genetic screening strategies. All large-scale trials studying the effect of ascorbic acid in Charcot-Marie-Tooth 1A have now been completed and were negative. Efforts have been made to design more robust outcome-measures for clinical trials. Promising results with lonaprisan, curcumin and histone deacetylase 6 inhibitors have been obtained in animal models.Charcot-Marie-Tooth is the most common form of inherited peripheral neuropathy and represents the most prevalent hereditary neuromuscular disorder. The genetic spectrum spans more than 70 genes. Gene discovery has been revolutionized recently by new high-throughput molecular technologies. In addition, the phenotypic diversity has grown tremendously. This is a major challenge for geneticists and neurologists. No effective therapy is available for Charcot-Marie-Tooth. Several large trials with ascorbic acid were negative but research into novel compounds continues.

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[55]
Rauchenzauner M,Fruhwirth M,Hecht M,et al.A novel variant in the HINT1 gene in a girl with autosomal recessive axonal neuropathy with neuromyotonia:thorough neurological examination gives the clue[J].Neuropediatrics,2016,47(2):119-122.doi:10.1055/s-0035-1570493.We report a girl with autosomal recessive axonal neuropathy with neuromyotonia (ARAN-NM) who presented with asymmetric gait impairment, foot drop, and action myotonia on fast handgrip. Electrophysiological studies showed symmetrical axonal motor greater than sensory neuropathy, and neuromyotonic discharges on needle electromyography. ARAN-NM was confirmed by molecular genetic testing, which revealed a novel homozygous missense variant c.100G65>65A [p.(Glu34Lys)] in HINT1. This case shows that the diagnosis of ARAN-NM, as a new entity, has to be considered in the differential diagnosis of polyneuropathy in combination with neuromyotonia/action myotonia in children, even with asymmetric clinical presentation.

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[56]
Zimon M,Baets J,Almeida-Souza L,et al.Loss-of-function mutations in HINT1 cause axonal neuropathy with neuromyotonia[J].Nat Genet,2012,44(10):1080-1083.doi:10.1038/ng.2406.Inherited peripheral neuropathies are frequent neuromuscular disorders known for their clinical and genetic heterogeneity. In 33 families, we identified 8 mutations in HINT1 (encoding histidine triad nucleotide-binding protein 1) by combining linkage analyses with next-generation sequencing and subsequent cohort screening of affected individuals. Our study provides evidence that loss of functional HINT1 protein results in a distinct phenotype of autosomal recessive axonal neuropathy with neuromyotonia.

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[57]
Maddison P.Neuromyotonia[J].Clin Neurophysiol,2006,117(10):2118-2127. doi:10.1016/j.clinph.2006.03.008.

[58]
Zimon M,Battaloglu E,Parman Y,et al.Unraveling the genetic landscape of autosomal recessive Charcot-Marie-Tooth neuropathies using a homozygosity mapping approach[J].Neurogenetics,2015,16(1):33-42.doi:10.1007/s10048-014-0422-0.Autosomal recessive forms of Charcot-Marie-Tooth disease (ARCMT) are rare but severe disorders of the peripheral nervous system. Their molecular basis is poorly understood due to the extensive genetic and clinical heterogeneity, posing considerable challenges for patients, physicians, and researchers. We report on the genetic findings from a systematic study of a large collection of 174 independent ARCMT families. Initial sequencing of the three most common ARCMT genes ( ganglioside-induced differentiation protein 1 — GDAP1 , SH3 domain and tetratricopeptide repeats-containing protein 2 — SH3TC2 , histidine-triad nucleotide binding protein 1 — HINT1 ) identified pathogenic mutations in 41 patients. Subsequently, 87 selected nuclear families underwent single nucleotide polymorphism (SNP) genotyping and homozygosity mapping, followed by targeted screening of known ARCMT genes. This strategy provided molecular diagnosis to 2202% of the families. Altogether, our unbiased genetic approach identified pathogenic mutations in ten ARCMT genes in a total of 41.302% patients. Apart from a newly described founder mutation in GDAP1 , the majority of variants constitute private molecular defects. Since the gene testing was independent of the clinical phenotype of the patients, we identified mutations in patients with unusual or additional clinical features, extending the phenotypic spectrum of the SH3TC2 gene. Our study provides an overview of the ARCMT genetic landscape and proposes guidelines for tackling the genetic heterogeneity of this group of hereditary neuropathies.

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[59]
Seburn KL,Morelli KH,Jordanova A,et al.Lack of neuropathy-related phenotypes in hint1 knockout mice[J].J Neuropathol Exp Neurol,2014,73(7):693-701.doi:10.1097/nen.0000000000000085.Mutations in HINT1, the gene encoding histidine triad nucleotide-binding protein 1 (HINT1), cause a recessively inherited peripheral neuropathy that primarily involves motor dysfunction and is usually associated with neuromyotonia (i.e. prolonged muscle contraction resulting from hyperexcitability of peripheral nerves). Because these mutations are hypothesized to cause loss of function, we analyzed Hint1 knockout mice for their relevance as a disease model. Mice lacking Hint1 appeared normal and yielded normal behavioral test results or motor performance, although they moved more slowly and for a smaller fraction of time in an open-field arena than wild-type mice. Muscles, neuromuscular junctions, and nodes of Ranvier were anatomically normal and did not show evidence of degeneration or regeneration. Axon numbers and myelination in peripheral nerves were normal at ages 4 and 13 months. Axons were slightly smaller than those in wild-type mice at age 4 months, but this did not cause a decrease in conduction velocity, and no differences in axon diameters were detected at 13 months. With electromyography, we were unable to detect neuromyotonia even after using supraphysiologic stimuli and stressors such as reduced temperature or 3,4-diaminopyridine to block potassium channels. Therefore, we conclude that Hint1 knockout mice may be useful for studying the biochemical activities of HINT1, but these mice do not provide a disease model or a means for investigating the basis of HINT1-associated neuropathy and neuromyotonia.

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[60]
Horga A,Cottenie E,Tomaselli PJ,et al.Absence of HINT1 mutations in a UK and Spanish cohort of patients with inherited neuropathies[J].J Neurol,2015,262(8):1984-1986.doi:10.1007/s00415-015-7851-z.Biallelic mutations in the HINT1 gene were recently identified as the cause of axonal neuropathy with neuromyotonia. It has been suggested that HINT1 mutations may indeed account for 11% of all inherited neuropathies with autosomal recessive inheritance. However, 81% of patients HINT 1-related neuropathies reported to date are originally from five European countries and the global prevalence of the disorder is still unknown. In our study, we aimed to determine the frequency of HINT1 mutations by direct sequencing in a cohort of 152 patients with inherited neuropathies from the UK and Spain, where no cases have been described to date. We failed to identify patients with clinical myotonia, neuromyotonia or pathogenic mutations in HINT1 . Our results support that HINT1 -related neuropathies are not homogeneously distributed among European populations, which may be explained by founder effects. This geographical variability also underlines the importance of considering the ethnic background when screening for mutations in neuropathy-related genes.

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[61]
Jerath NU,Shy ME,Grider T,et al.A case of neuromyotonia and axonal motor neuropathy:A report of a HINT1 mutation in the United States[J].Muscle Nerve,2015,52(6):1110-1113.doi:10.1002/mus.24774.ABSTRACT Introduction: HINT1 mutations cause an autosomal recessive distal hereditary motor axonal neuropathy with neuromyotonia. This is a case report of a HINT1 mutation in the United States. Methods: A 30-year-old man of Slovenian heritage and no significant family history presented with scoliosis as a child and later developed neuromyotonia and distal weakness. Electrodiagnostic testing revealed an axonal motor neuropathy and neuromyotonic discharges. Previous diagnostic work-up, including testing for Cx32 , MPZ , PMP-22 , NF-L , EGR2 , CLCN1 , DM1 , DM2 , SMN exon 7/8, emerin, LMNA , MPK , SCNA4 , acid maltase gene, paraneoplastic disorder, and a sural nerve biopsy, was negative. Results: Genetic testing for a HINT1 mutation was performed and revealed a homozygous mutation at p.Arg37Pro. Conclusion: This entity should be distinguished clinically and genetically from myotonic dystrophy and channelopathies with the clinical features of neuromyotonia and an axonal neuropathy. This case illustrates the importance of identifying the correct phenotype to avoid unnecessary and costly evaluations. Muscle Nerve 52: 11101113, 2015

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[62]
Lassuthova P,Brozkova DS,Krutova M,et al.Mutations in HINT1 are one of the most frequent causes of hereditary neuropathy among Czech patients and neuromyotonia is rather an underdiagnosed symptom[J].Neurogenetics,2015,16(1):43-54.doi:10.1007/s10048-014-0427-8.Mutations in the HINT1 gene were recently discovered as being the major cause of autosomal recessive axonal neuropathy with neuromyotonia. This combination was clinically recognized and described prev

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[63]
Elashoff M,Higgs BW,Yolken RH,et al.Meta-analysis of 12 genomic studies in bipolar disorder[J].J Mol Neurosci,2007,31(3):221-243.Multiple genome-wide expression studies of bipolar disorder have been published. However, a unified picture of the genomic basis for the disease has not yet emerged. Genes identified in one study often fail to be identified in other studies, prompting the question of whether microarray studies in the brain are inherently unreliable. To answer this question, we performed a meta-analysis of 12 microarray studies of bipolar disorder. These studies included >500 individual array samples, on a range of microarray platforms and brain regions. Although we confirmed that individual studies showed some differences in results, clear and striking regulation patterns emerged across the studies. These patterns were found at the individual gene level, at the functional level, and at the broader pathway level. The patterns were generally found to be reproducible across platform and region, and were highly statistically significant. We show that the seeming discordance between the studies was primarily a result of the following factors, which are also typical for other brain array studies: (1) Sample sizes were, in retrospect, too small; (2) criteria were at once too restrictive (generally focusing on fold changes >1.5) and too broad (generally using p <0.05 or p <0.01 as criteria for significance); and (3) statistical adjustments were not consistently applied for confounders. In addition to these general conclusions, we also summarize the primary biological findings of the meta-analysis, focusing on areas that confirm previous research and also on novel findings.

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[64]
Barbier E,Wang JB.Anti-depressant and anxiolytic like behaviors in PKCI/HINT1 knockout mice associated with elevated plasma corticosterone level[J].BMC Neurosci,2009,10:132.doi:10.1186/1471-2202-10-132.Background Protein kinase C interacting protein (PKCI/HINT1) is a small protein belonging to the histidine triad (HIT) family proteins. Its brain immunoreactivity is located in neurons and neuronal processes. PKCI/HINT1 gene knockout (KO) mice display hyper-locomotion in response to D-amphetamine which is considered a positive symptom of schizophrenia in animal models. Postmortem studies identified PKCI/HINT1 as a candidate molecule for schizophrenia and bipolar disorder. We investigated the hypothesis that the PKCI/HINT1 gene may play an important role in regulating mood function in the CNS. We submitted PKCI/HINT1 KO mice and their wild type (WT) littermates to behavioral tests used to study anti-depressant, anxiety like behaviors, and goal-oriented behavior. Additionally, as many mood disorders coincide with modifications of hypothalamic-pituitary-adrenal (HPA) axis function, we assessed the HPA activity through measurement of plasma corticosterone levels. Results Compared to the WT controls, KO mice exhibited less immobility in the forced swim (FST) and the tail suspension (TST) tests. Activity in the TST tended to be attenuated by acute treatment with valproate at 300 mg/kg in KO mice. The PKCI/HINT1 KO mice presented less thigmotaxis in the Morris water maze and spent progressively more time in the lit compartment in the light/dark test. In a place navigation task, KO mice exhibited enhanced acquisition and retention. Furthermore, the afternoon basal plasma corticosterone level in PKCI/HINT1 KO mice was significantly higher than in the WT. Conclusion PKCI/HINT1 KO mice displayed a phenotype of behavioral and endocrine features which indicate changes of mood function, including anxiolytic-like and anti-depressant like behaviors, in conjunction with an elevated corticosterone level in plasma. These results suggest that the PKCI/ HINT 1 gene could be important for the mood regulation function in the CNS.

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[65]
Martins-de-Souza D,Guest PC,Harris LW,et al. Identification of proteomic signatures associated with depression and psychotic depression in post-mortem brains from major depression patients[J].Transl Psychiatry,2012,2:e87.doi:10.1038/tp.2012.13.Major depressive disorder (MDD) is a leading cause of disability worldwide and results tragically in the loss of almost one million lives in Western societies every year. This is due to poor understanding of the disease pathophysiology and lack of empirical medical tests for accurate diagnosis or for guiding antidepressant treatment strategies. Here, we have used shotgun proteomics in the analysis ofpost-mortemdorsolateral prefrontal cortex brain tissue from 24 MDD patients and 12 matched controls. Brain proteomes were pre-fractionated by gel electrophoresis and further analyzed by shotgun data-independent label-free liquid chromatography-mass spectrometry. This led to identification of distinct proteome fingerprints between MDD and control subjects. Some of these differences were validated by Western blot or selected reaction monitoring mass spectrometry. This included proteins associated with energy metabolism and synaptic function and we also found changes in the histidine triad nucleotide-binding protein 1 (HINT1), which has been implicated recently in regulation of mood and behavior. We also found differential proteome profiles in MDD with (n=11) and without (n=12) psychosis. Interestingly, the psychosis fingerprint showed a marked overlap to changes seen in the brain proteome of schizophrenia patients. These findings suggest that it may be possible to contribute to the disease understanding by distinguishing different subtypes of MDD based on distinct brain proteomic profiles.

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[66]
Ge L,Zhu MM,Yang JY,et al.Differential proteomic analysis of the anti-depressive effects of oleamide in a rat chronic mild stress model of depression[J].Pharmacol Biochem Behav,2015,131:77-86.doi:10.1016/j.pbb. 2015.01.017.Abstract Depression is a complex psychiatric disorder, and its etiology and pathophysiology are not completely understood. Depression involves changes in many biogenic amine, neuropeptide, and oxidative systems, as well as alterations in neuroendocrine function and immune-inflammatory pathways. Oleamide is a fatty amide which exhibits pharmacological effects leading to hypnosis, sedation, and anti-anxiety effects. In the present study, the chronic mild stress (CMS) model was used to investigate the antidepressant-like activity of oleamide. Rats were exposed to 10weeks of CMS or control conditions and were then subsequently treated with 2weeks of daily oleamide (5mg/kg, i.p.), fluoxetine (10mg/kg, i.p.), or vehicle. Protein extracts from the hippocampus were then collected, and hippocampal maps were generated by way of two-dimensional gel electrophoresis (2-DE). Altered proteins induced by CMS and oleamide were identified through mass spectrometry and database searches. Compared to the control group, the CMS rats exhibited significantly less body weight gain and decreased sucrose consumption. Treatment with oleamide caused a reversal of the CMS-induced deficit in sucrose consumption. In the proteomic analysis, 12 protein spots were selected and identified. CMS increased the levels of adenylate kinase isoenzyme 1 (AK1), nucleoside diphosphate kinase B (NDKB), histidine triad nucleotide-binding protein 1 (HINT1), acyl-protein thioesterase 2 (APT-2), and glutathione S-transferase A4 (GSTA4). Compared to the CMS samples, seven spots changed significantly following treatment with oleamide, including GSTA4, glutathione S-transferase A6 (GSTA6), GTP-binding nuclear protein Ran (Ran-GTP), ATP synthase subunit d, transgelin-3, small ubiquitin-related modifier 2 (SUMO2), and eukaryotic translation initiation factor 5A-1 (eIF5A1). Of these seven proteins, the level of eIF5A1 was up-regulated, whereas the remaining proteins were down-regulated. In conclusion, oleamide has antidepressant-like properties in the CMS rat model. The identification of proteins altered by CMS and oleamide treatment provides support for targeting these proteins in the development of novel therapies for depression. Copyright © 2015. Published by Elsevier Inc.

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[67]
Varadarajulu J,Lebar M,Krishnamoorthy G,et al.Increased anxiety-related behaviour in Hint1 knockout mice[J].Behav Brain Res,2011,220(2):305-311. doi:10.1016/j.bbr.2011.02.012.Several reports have implicated a role for the histidine triad nucleotide-binding protein-1 (Hint1) in psychiatric disorders. We have studied the emotional behaviour of male Hint1 knockout (Hint1 KO) mice in a battery of tests and performed biochemical analyses on brain tissue. The behavioural analysis revealed that Hint1 KO mice exhibit an increased emotionality phenotype compared to wildtype (WT) mice, while no significant differences in locomotion or general exploratory activity were noted. In the elevated plus-maze (EPM) test, the Hint1 KO animals entered the open arms of the apparatus less often than WT littermates. Similarly, in the dark–light box test, Hint1 KO mice spent less time in the lit compartment and the number of entries were reduced, which further confirmed an increased anxiety-related behaviour. Moreover, the Hint1 KO animals showed significantly more struggling and less floating behaviour in the forced swim test (FST), indicating an increased emotional arousal in aversive situations. Hint1 is known as a protein kinase C (PKC) interacting protein. Western blot analysis showed that PKCγ expression was elevated in Hint1 KO compared to WT mice. Interestingly, PKCγ mRNA levels of the two groups did not show a significant difference, implying a post-transcriptional PKCγ regulation. In addition, PKC enzymatic activity was increased in Hint1 KO compared to WT mice. In summary, our results indicate a role for Hint1 and PKCγ in modulating anxiety-related and stress-coping behaviour in mice.

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[68]
Jackson KJ,Wang JB,Barbier E,et al.Acute behavioral effects of nicotine in male and female HINT1 knockout mice[J].Genes Brain Behav,2012,11(8):993-1000.doi:10.1111/j.1601-183X.2012.00827.x.Human genetic association and brain expression studies, and mouse behavioral and molecular studies implicate a role for the histidine triad nucleotide-binding protein 1 (HINT1) in schizophrenia, bipolar disorder, depression and anxiety. The high comorbidity between smoking and psychiatric disorders, schizophrenia in particular, is well established. Associations with schizophrenia and HINT1 are also sex specific, with effects more predominant in males; however, it is unknown if sex differences associated with the gene extend to other phenotypes. Thus, in this study, using a battery of behavioral tests, we elucidated the role of HINT1 in acute nicotine-mediated behaviors using male and female HINT1 wild-type (+/+) and knockout (090808/090808) mice. The results show that male HINT1 090808/090808 mice were less sensitive to acute nicotine-induced antinociception in the tail-flick, but not hot-plate test. At low nicotine doses, male and female HINT1 090808/090808 mice were less sensitive to nicotine-induced hypomotility, although the effect was more pronounced in females. Baseline differences in locomotor activity observed in male HINT1 +/+ and 090808/090808 mice were absent in females. Nicotine did not produce an anxiolytic effect in male HINT1 090808/090808 mice, but rather an anxiogenic response. Diazepam also failed to induce an anxiolytic response in these mice, suggesting a general anxiety phenotype not specific to nicotine. Differences in anxiety-like behavior were not observed in female mice. These results further support a role for HINT1 in nicotine-mediated behaviors and suggest that alterations in the gene may have differential effects on phenotype in males and females.

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[69]
Liu F,Ma J,Liu P,et al.Hint1 gene deficiency enhances the supraspinal nociceptive sensitivity in mice[J].Brain Behav,2016,6(8):e00496.doi:10.1002/brb3.496.Abstract IntroductionPrevious studies have indicated a possible role of histidine triad nucleotide-binding protein 1 (HINT1) on sustaining the regulatory crosstalk of N-methyl-D-aspartate acid glutamate receptors (NMDARs) and G-protein-coupled receptors (GPCRs) such as the μ -opioid receptor (MOR). Both receptors are present in the midbrain periaqueductal gray neurons, an area that plays a central role in the supraspinal antinociceptive process. MethodsIn the present study, a battery of pain-related behavioral experiments was applied to Hint1 knockout, heterozygous and wild-type mice. Both the male and female mice were investigated to assess the differences between genders. Results Hint1 61/61 mice presented significant shorter latency at 50°C in both male and female in hot plate test while no significant difference was found in tail filck test. In Von Frey hairs test Hint1 61/61 mice were more sensitive than Hint1 +/+ mice, presenting a lower withdrawal threshold and enhanced relative frequency of paw withdrawal. The average flinches and licking time of Hint1 61/61 mice were more than that of Hint1 +/+ mice in formalin test. ConclusionThe02absence of Hint1 gene-enhanced supraspinal nociceptive sensitivity in mice, including thermal, mechanical and inflammatory hyperalgesia. Meanwhile, there was no certain evidence indicating the haploinsufficiency and gender differences of Hint1 gene in pain-related behaviors.

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[70]
Vicente-Sanchez A,Sanchez-Blazquez P,Rodriguez-Munoz M,et al.HINT1 protein cooperates with cannabinoid 1 receptor to negatively regulate glutamate NMDA receptor activity[J].Mol Brain,2013,6:42.doi:10.1186/1756-6606-6-42.

[71]
Sanchez-Blazquez P,Rodriguez-Munoz M,Berrocoso E,et al.The plasticity of the association between mu-opioid receptor and glutamate ionotropic receptor N in opioid analgesic tolerance and neuropathic pain[J].Eur J Pharmacol,2013,716(1-3):94-105.doi:10.1016/j.ejphar.2013.01.066.Multiple groups have reported the functional cross-regulation between mu-opioid (MOP) receptor and glutamate ionotropic receptor N (GluN), and the post-synaptic association of these receptors has been implicated in the transmission and modulation of nociceptive signals. Opioids, such as morphine, disrupt the MOP receptor–GluN receptor complex to stimulate the activity of GluN receptors via protein kinase C (PKC)/Src. This increased GluN receptor activity opposes MOP receptor signalling, and via neural nitric oxide synthase (nNOS) and calcium and calmodulin regulated kinase II (CaMKII) induces the phosphorylation and uncoupling of the opioid receptor, which results in the development of morphine analgesic tolerance. Both experimental in vivo activation of GluN receptors and neuropathic pain separate the MOP receptor–GluN receptor complex via protein kinase A (PKA) and reduce the analgesic capacity of morphine. The histidine triad nucleotide-binding protein 1 (HINT1) associates with the MOP receptor C-terminus and connects the activities of MOP receptor and GluN receptor. In HINT1 (61/61) mice, morphine promotes enhanced analgesia and produces tolerance that is not related to GluN receptor activity. In these mice, the GluN receptor agonist N -methyl- d -aspartate acid (NMDA) does not antagonise the analgesic effects of morphine. Treatments that rescue morphine from analgesic tolerance, such as GluN receptor antagonism or PKC, nNOS and CaMKII inhibitors, all induce MOP receptor–GluN receptor re-association and reduce GluN receptor/CaMKII activity. In mice treated with NMDA or suffering from neuropathic pain (induced by chronic constriction injury, CCI), GluN receptor antagonists, PKA inhibitors or certain antidepressants also diminish CaMKII activity and restore the MOP receptor–GluN receptor association. Thus, the HINT1 protein stabilises the association between MOP receptor and GluN receptor, necessary for the analgesic efficacy of morphine, and this coupling is reduced following the activation of GluN receptors, similar to what is observed in neuropathic pain.

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[72]
Garzon J,Herrero-Labrador R,Rodriguez-Munoz M,et al.HINT1 protein:a new therapeutic target to enhance opioid antinociception and block mechanical allodynia[J].Neuropharmacology,2015,89:412-423.doi:10.1016/j. neuropharm.2014.10.022.Abstract In the nervous system, the glutamate N-methyl-D-aspartate receptor (NMDAR) restricts the activity of the mu-opioid receptor (MOR). Both receptors are present in midbrain periaqueductal grey (PAG) neurons, an area that plays a central role in the supraspinal antinociceptive effects of opioids. The cross-talk that occurs between these receptors is sustained by the MOR-associated histidine triad nucleotide binding protein 1 (HINT1), which displays nucleoside phosphoramidase and acyl-AMP hydrolase activity. Here we report that the inhibitor of HINT1 enzymatic activity guanosine-5-tryptamine carbamate (TpGc) significantly enhanced morphine antinociception while preventing the development of tolerance. At the molecular level, TpGc reduced the capacity of MORs to recruit NMDAR activity to negatively regulate opioid signaling. In mice suffering from chronic constriction injury concurrent with increased NMDAR activity, a single intracerebroventricular administration of TpGc attenuated NMDAR function and alleviated mechanical allodynia for several days. These data suggest a potential therapeutic role for HINT1 inhibitors in the clinical management of acute and neuropathic pain.

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[73]
Klepstad P,Fladvad T,Skorpen F,et al.Influence from genetic variability on opioid use for cancer pain:a European genetic association study of 2294 cancer pain patients[J].Pain,2011,152(5):1139-1145.doi:10.1016/j.pain.2011.01.040.None of 112 SNPs in the 25 candidate genes OPRM1 , OPRD1 , OPRK1 , ARRB2 , GNAZ , HINT1 , Stat6 , ABCB1 , COMT , HRH1 , ADRA2A , MC1R , TACR1 , GCH1 , DRD2 , DRD3 , HTR3A , HTR3B , HTR2A , HTR3C , HTR3D , HTR3E , HTR1 , or CNR1 showed significant associations with opioid dose in both the development and the validation analyzes. These findings do not support the use of pharmacogenetic analyses for the assessed SNPs to guide opioid treatment. The study also demonstrates the importance of validating findings obtained in genetic association studies to avoid reporting spurious associations as valid findings. To elicit knowledge about new genes that influence pain and the need for opioids, strategies other than the candidate gene approach is needed.

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[74]
Jackson KJ,Chen Q,Chen J,et al.Association of the histidine-triad nucleotide-binding protein-1(HINT1) gene variants with nicotine dependence[J].Pharmacogenomics J,2011,11(4):251-257.doi:10.1038/tpj.2010.41.Abstract The histidine triad nucleotide-binding protein-1 gene (HINT1) is implicated in schizophrenia and in the behavioral effects of morphine and amphetamine. Because nicotine dependence (ND) is highly comorbid with schizophrenia and other substance abuse, we examined the association of HINT1 with ND. Association analyses from two independent samples show that HINT1 gene variants are associated with ND phenotypes. Furthermore, human postmortem mRNA expression shows that smoking status and genotype influence HINT1 expression in the brain. In animal studies, western blot analyses show an increase of HINT1 protein level in the mouse nucleus accumbens (NAc) after chronic nicotine exposure. This increase was reduced after treatment with the nicotinic-receptor antagonist mecamylamine, and 24 and 72/h after cessation of nicotine treatment. These results indicate a genetic association between HINT1 variants and ND, and indicate that nicotine-induced modulation of HINT1 level may be involved in mechanisms of excess smoking.

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[75]
Fang J,Wang X,He B.Association between common genetic variants in the opioid pathway and smoking behaviors in Chinese men[J].Behav Brain Funct,2014,10:2.doi:10.1186/1744-9081-10-2.Background There is biological evidence that the brain opioidergic system plays a critical role in the addictive properties of nicotine. The purpose of the present study was to examine the associations of single nucleotide polymorphisms (SNPs) in the genes encoding mu-opioid receptor (MOR) and the MOR-interacting proteins (including OPRM1, ARRB2, and HINT1) with smoking behaviors in Chinese men. Methods A total of 284 subjects (including current and ex-smokers) were recruited. Special questionnaires were used to assess smoking behaviors including age of smoking initiation, daily cigarette consumption, and Fagerstr??m test for nicotine dependence (FTND) score. Participant samples were genotyped for six SNPs in the opioid pathway genes: rs1799971 in OPRM1, rs1045280, rs2036657 and rs3786047 in ARRB2, rs3852209 and rs2278060 in HINT1. Linear and logistic regression models were used to determine single-locus and haplotype-based association analyses. Results There was no significant association between any of SNPs analyzed and smoking behaviors. Logistic regression analyses under dominant, recessive, and additive models showed no significant associations of the six SNPs with smoking status (current vs. ex-smokers). After adjustment for age at enrollment and smoking initiation age, HINT1 rs3852209 was significantly associated with smoking status with an OR of 0.54 (95% CI, 0.31-0.95; P???=???0.03) under dominant inheritance model. No haplotypes in ARRB2 or HINT1 were related to smoking status. Conclusions The present study indicates no significant association between common genetic variations in MOR and MOR-interacting proteins and smoking behaviors in Chinese men, and gives suggestive evidence that HINT1 rs3852209 may be related to smoking status. The findings require confirmation from further studies in additional larger samples.

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[76]
Ray R,Jepson C,Wileyto EP,et al.Genetic variation in mu-opioid-receptor-interacting proteins and smoking cessation in a nicotine replacement therapy trial[J].Nicotine Tob Res,2007,9(11):1237-1241.doi:10.1080/14622200701648367.

[77]
Jackson KJ,Wang JB,Barbier E,et al.The histidine triad nucleotide binding 1 protein is involved in nicotine reward and physical nicotine withdrawal in mice[J].Neurosci Lett,2013,550:129-133.doi:10.1016/j.neulet. 2013.06.027.Abstract Smoking rates among individuals with schizophrenia are significantly higher than the general population. One possible explanation for this comorbidity is that there are shared genes and biological pathways between smoking and schizophrenia. The histidine triad nucleotide binding protein 1 (HINT1) is a potential candidate, as genetic association and expression studies implicate the gene in both schizophrenia and nicotine dependence; however, the behavioral role of HINT1 in nicotine dependence is unknown. Thus, the goal of the current study was to determine the behavioral role of HINT1 in nicotine dependence. We tested male HINT1 wild-type (+/+) and knockout (-/-) mice in the nicotine conditioned place preference (CPP) test of reward, a nicotine withdrawal model assessing both physical and affective signs, and the nicotine withdrawal conditioned place aversion (CPA) test. HINT1 -/- mice failed to develop a significant nicotine CPP and physical withdrawal signs (hyperalgesia and somatic signs) were attenuated in HINT1 -/- mice. Conversely, HINT1 -/- mice developed a significant nicotine withdrawal CPA similar to their ++ counterparts. Overall, our data support a role for the HINT1 gene in mediating behaviors associated with nicotine reward and physical nicotine withdrawal, and provide insight into the role of HINT1 in nicotine dependence-like behaviors. Copyright © 2013. Published by Elsevier Ireland Ltd.

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[78]
Romanova EV,Lee JE,Kelleher NL,et al.Mass spectrometry screening reveals peptides modulated differentially in the medial prefrontal cortex of rats with disparate initial sensitivity to cocaine[J].Aaps J,2010,12(3):443-454.doi:10.1208/s12248-010-9204-2.To better understand why certain individuals are more vulnerable to cocaine abuse and addiction, we identify peptide markers associated with individual variation in sensitivity to the behavioral effects of cocaine. Previous studies in rats show that low, compared to high, cocaine responders are more sensitive to cocaine-induced behavioral plasticity (sensitization), exhibit enhanced conditioning to cocaine rewarding effects, and are more motivated to self administer cocaine. In the current study, we combine matrix-assisted laser desorption/ionization mass spectrometry with multivariate statistical methods to analyze tissue extracts from rat dorsal striatum, nucleus accumbens, and medial prefrontal cortex (mPFC) to examine trends in peptide changes that coincide with behavioral phenotype. Peptide profiles of these three regions from individual animals were characterized via mass spectrometry. Resulting mass peaks that were statistically different between these groups were identified using principal component analysis. The mass peaks were then identified in pooled samples via multistage liquid chromatography mass spectrometry. A total of 74 peptides from 28 proteins were sequenced from defined brain regions. Statistically significant changes in peak intensities for seven peptides were found in the mPFC of rats given a single injection of 10 mg/kg cocaine, with low cocaine responders showing 2-fold increase in peak intensities for the acetylated N terminus peptides of stathmin and Hint 1, as well as truncated ATP synthase. These results suggest that distinct peptide profiles in the mPFC are associated with individuals that exhibit reduced sensitivity to the behavioral effects of cocaine.

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[79]
Sabeti J,Gerhardt GA,Zahniser NR.Individual differences in cocaine-induced locomotor sensitization in low and high cocaine locomotor-responding rats are associated with differential inhibition of dopamine clearance in nucleus accumbens[J].J Pharmacol Exp Ther,2003,305(1):180-190.doi:10.1124/jpet.102.047258.

[80]
Klein DA,Gulley JM.Reduced sensitivity to the locomotor-stimulant effects of cocaine is associated with increased sensitivity to its discriminative stimulus properties[J].Behav Pharmacol,2009,20(1):67-77.doi:10.1097/FBP.0b013e3283242fdd.Abstract Outbred Long-Evans rats exhibit wide variation in their locomotor response to cocaine. Here, we investigated the relationship between these individual differences and interoceptive effects of cocaine in low cocaine responder (LCR) and high cocaine responder (HCR) phenotypes. Rats were trained to discriminate cocaine (10.0 mg/kg, intraperitoneally) from saline by repeated pairings of injections with one of two response levers. In subsequent tests for stimulus generalization to other cocaine doses (1.25-15.0 mg/kg), LCRs exhibited partial-to-full generalization at 1.85 and 2.5 mg/kg cocaine, respectively, whereas HCRs did not. When the selective 5-HT reuptake inhibitor fluoxetine (5.0 mg/kg) was coadministered with saline or different cocaine doses, we observed similar upward shifts in dose-response in both phenotypes. In contrast, coadministration of the 5-HT2A/2C agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 0.3 mg/kg) led to partial substitution of DOI for cocaine and enhancement of the stimulus properties of 1.25 mg/kg cocaine in LCRs only. Finally, a retest of cocaine-induced locomotion after discrimination testing revealed marked behavioral sensitization in LCRs and modest changes in behavior in HCRs. Taken together, these results suggest that initial sensitivity to the locomotor-stimulant effects of cocaine is inversely related to its interoceptive properties and that differences in 5-HT systems may contribute to the phenotypic differences observed.

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[81]
Allen RM,Everett CV,Nelson AM,et al.Low and high locomotor responsiveness to cocaine predicts intravenous cocaine conditioned place preference in male Sprague-Dawley rats[J].Pharmacol Biochem Behav,2007,86(1):37-44.doi:10.1016/j.pbb.2006.12.005.Outbred, male Sprague awley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively) based on cocaine-induced locomotor activity in an open-field arena. This difference reflects cocaine's ability to inhibit the striatal dopamine transporter and predicts development of sensitization. To investigate the relationship between initial cocaine locomotor responsiveness and cocaine reward, here we first classified rats as either LCRs or HCRs in a conditioned place preference (CPP) apparatus. Subsequently, we conducted cocaine conditioning trials, twice-daily over 4days with vehicle and cocaine (10mg/kg, i.p. or 1mg/kg, i.v.). When cocaine was administered by the i.p. route, similar to previous findings in the open-field, LCRs and HCRs were readily classified and locomotor sensitization developed in LCRs, but not HCRs. However, cocaine CPP was not observed. In contrast, when cocaine was administered by the i.v. route, the LCR/HCR classification not only predicted sensitization, but also CPP, with only LCR rats exhibiting sensitization and cocaine conditioning. Our findings show that the initial locomotor response to cocaine can predict CPP in male Sprague awley rats under conditions when place conditioning develops, and that LCRs may be more prone to develop conditioning in the context of cocaine reward.

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[82]
Mandt BH,Schenk S,Zahniser NR,et al.Individual differences in cocaine-induced locomotor activity in male Sprague-Dawley rats and their acquisition of and motivation to self-administer cocaine[J].Psychopharmacology(Berl),2008,201(2):195-202.doi:10.1007/s00213-008-1265-x.Factors that increase an individual's susceptibility to cocaine dependence remain largely unknown. We have previously shown that adult outbred male Sprague-Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively) based on their locomotor activity following the administration of a single dose of cocaine (10 mg/kg, i.p.). Furthermore, LCR/HCR classification predicts dopamine transporter function/inhibition, cocaine-induced locomotor sensitization, and cocaine-conditioned place preference.The present study assessed LCR/HCR classification and the development of locomotor sensitization on the latency to acquire cocaine self-administration and motivation to self-administer cocaine.LCRs and HCRs did not differ in their latency to acquire low-dose cocaine self-administration (0.25 mg/kg/infusion over 12 s, fixed ratio 1 schedule of reinforcement). In a follow-up experiment, repeated experimenter-administered injections of cocaine (10 mg/kg, i.p.) resulted in locomotor sensitization for LCRs, but not HCRs; nonetheless, all rats exhibited decreased latency to acquire cocaine self-administration compared to the first experiment. Repeated cocaine preexposure and LCR/HCR classification predicted break point when rats responded for cocaine under a progressive ratio schedule of reinforcement (0.25, 0.5, and 1.0 mg/kg/infusion; multiple exposure>single exposure, LCR>HCR), but there was no interaction between these variables.Although LCR/HCR classification did not predict the rate of acquisition of cocaine self-administration under these conditions, LCR rats demonstrated greater responding for cocaine after acquisition (PR). Thus, these findings demonstrate the relevance of using the LCR/HCR model when studying susceptibility to cocaine dependence.

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[83]
Abdel Rassoul R,Alves S,Pantesco V,et al.Distinct transcriptome expression of the temporal cortex of the primate Microcebus murinus during brain aging versus Alzheimer’s disease-like pathology[J].PLoS One,2010,5(9).doi:10.1371/journal.pone.0012770.Abstract Aging is the primary risk factor of neurodegenerative disorders such as Alzheimer's disease (AD). However, the molecular events occurring during brain aging are extremely complex and still largely unknown. For a better understanding of these age-associated modifications, animal models as close as possible to humans are needed. We thus analyzed the transcriptome of the temporal cortex of the primate Microcebus murinus using human oligonucleotide microarrays (Affymetrix). Gene expression profiles were assessed in the temporal cortex of 6 young adults, 10 healthy old animals and 2 old, "AD-like" animals that presented -amyloid plaques and cortical atrophy, which are pathognomonic signs of AD in humans. Gene expression data of the 14,911 genes that were detected in at least 3 samples were analyzed. By SAM (significance analysis of microarrays), we identified 47 genes that discriminated young from healthy old and "AD-like" animals. These findings were confirmed by principal component analysis (PCA). ANOVA of the expression data from the three groups identified 695 genes (including the 47 genes previously identified by SAM and PCA) with significant changes of expression in old and "AD-like" in comparison to young animals. About one third of these genes showed similar changes of expression in healthy aging and in "AD-like" animals, whereas more than two thirds showed opposite changes in these two groups in comparison to young animals. Hierarchical clustering analysis of the 695 markers indicated that each group had distinct expression profiles which characterized each group, especially the "AD-like" group. Functional categorization showed that most of the genes that were up-regulated in healthy old animals and down-regulated in "AD-like" animals belonged to metabolic pathways, particularly protein synthesis. These data suggest the existence of compensatory mechanisms during physiological brain aging that disappear in "AD-like" animals. These results open the way to new exploration of physiological and "AD-like" aging in primates.

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[84]
Weitzdoerfer R,Stolzlechner D,Dierssen M,et al.Reduction of nucleoside diphosphate kinase B,Rab GDP-dissociation inhibitor beta and histidine triad nucleotide-binding protein in fetal Down syndrome brain[J].J Neural Transm Suppl,2001(61):347-359.Information on the various factors leading to impairments in the developing brain of fetal Down Syndrome patients is limited to few histological reports. We therefore attempted to describe expression levels of proteins in brain using the proteomic technique of two-dimensional electrophoresis with subsequent mass spectroscopical identification of protein spots and quantification with specific software. Cortical tissue was obtained from autopsy of human fetal abortus. Protein levels of GTP-binding nuclear protein ran, guanine nucleotide-binding protein g(o), alpha subunit 2, guanine nucleotide-binding protein g(i)/g(s)/g(t) beta subunit 1, -beta subunit 2, guanine nucleotide-binding protein beta subunit 5, nucleoside diphosphate kinase A, nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta, Rho GDP-dissociation inhibitor 1, biphosphate 3-nucleotidase, small glutamine-rich tetra-tricopeptide repeat-containing protein and histidine triad nucleotide-binding protein were studied.Quantification revealed statistically significant reduced levels of nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta and histidine triad nucleotide-binding protein in fetal DS brain as compared to controls.We conclude that in early prenatal life proteins involved in neural differentiation, migration and synaptic transmission are impaired in DS cortex. These results may help to understand the abundant mechanisms leading to abnormalities in the wiring, structure and function of DS brain.

DOI

[85]
Krakowiak A,Pawlowska R,Kocon-Rebowska B,et al.Interactions of cellular histidine triad nucleotide binding protein 1 with nucleosides 5’-O-monophosphorothioate and their derivatives-Implication for desulfuration process in the cell[J].Biochim Biophys Acta,2014,1840(12):3357-3366.doi:10.1016/j.bbagen.2014.08.016.The intracellular Hint1 could be responsible for the in vivo desulfuration of nucleosides-5′-monophosphorothioate, thus it can contribute to the phosphorothioate oligonucleotides metabolism. H 2 S released during this process may participate in several physiological processes, thus NMPSs can be precursors/donors of H 2 S in vivo and can be used to study the effects of this gas in biological systems. Moreover, the controlled delivery of (d)NMPSs into cells may be of medicinal utility.

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[86]
di Masi A,Ascenzi P. H2S:a“double face”molecule in health and disease[J].Biofactors,2013,39(2):186-196.doi:10.1002/biof.1061.

[87]
Paul BD,Snyder SH.H2S:A novel gasotransmitter that signals by sulfhydration[J].Trends Biochem Sci,2015,40(11):687-700.doi:10.1016/j.tibs.2015.08.007.Hydrogen sulfide (H2S) is a gasotransmitter that signals via sulfhydration, a post-translational modification. Sulfhydration occurs on reactive cysteine residues and converts the Cys –SH group to an –SSH group. Sulfhydration modulates diverse physiological processes ranging from regulation of blood pressure to signaling in the nervous system. An emerging theme is the interplay of sulfhydration and nitrosylation that fine tunes signaling pathways. A variety of detection agents for H2S and sulfhydration have been developed to study the role of this modification in physiological systems.

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[88]
Rodriguez-Muñoz M,Sánchez-Blázquez P,Merlos M,et al.Endocannabinoid control of glutamate NMDA receptors:the therapeutic potential and consequences of dysfunction[J].Oncotarget,2016,7(34):55840-55862.doi:10.18632/oncotarget.10095.Abstract Glutamate is probably the most important excitatory neurotransmitter in the brain. The glutamate N-methyl-D-aspartate receptor (NMDAR) is a calcium-gated channel that coordinates with G protein-coupled receptors (GPCRs) to establish the efficiency of the synaptic transmission. Cross-regulation between these receptors requires the concerted activity of the histidine triad nucleotide-binding protein 1 (HINT1) and of the sigma receptor type 1 (1R). Essential brain functions like learning, memory formation and consolidation, mood and behavioral responses to exogenous stimuli depend on the activity of NMDARs. In this biological context, endocannabinoids are released to retain NMDAR activity within physiological limits. The efficacy of such control depends on HINT1/1R assisting in the physical coupling between cannabinoid type 1 receptors (CB1Rs) and NMDARs to dampen their activity. Subsequently, the calcium-regulated HINT1/1R protein tandem uncouples CB1Rs to prevent NMDAR hypofunction. Thus, early recruitment or a disproportionate cannabinoid induced response can bring about excess dampening of NMDAR activity, impeding its adequate integration with GPCR signaling. Alternatively, this control circuit can apparently be overridden in situations where bursts of NMDAR overactivity provoke convulsive syndromes. In this review we will discuss the possible relevance of the HINT1/1R tandem and its use by endocannabinoids to diminish NMDAR activity and their implications in psychosis/schizophrenia, as well as in NMDAR-mediated convulsive episodes.

DOI PMID

[89]
Sanchez-Blazquez P,Rodriguez-Munoz M,Garzon J.The cannabinoid receptor 1 associates with NMDA receptors to produce glutamatergic hypofunction:implications in psychosis and schizophrenia[J].Front Pharmacol,2014,4:169.doi:10.3389/fphar.2013.00169.The endocannabinoid system is widespread throughout the central nervous system and its type 1 receptor (CB1) plays a crucial role in preventing the neurotoxicity caused by activation of glutamate N-methyl-D-aspartate receptors (NMDARs). Indeed, it is the activity of NMDARs themselves that provides the demands on the endogenous cannabinoids in order to control their calcium currents. Therefore, a physiological role of this system is to maintain NMDAR activity within safe limits, thereby protecting neural cells from excitotoxicity. Thus, cannabinoids may be able to control NMDAR overactivation-related neural dysfunctions; however, the major obstacles to the therapeutic utilization of these compounds are their psychotropic effects and negative influence on cognitive performance. Studies in humans have indicated that abuse of smoked cannabis can promote psychosis and even circumstantially precipitate symptoms of schizophrenia, although the latter appears to require a prior vulnerability in the individual. It is possible that cannabinoids provoke psychosis/schizophrenia reflecting a mechanism common to neuroprotection: the reduction of NMDAR activity. Cannabinoids are proposed to produce such effect by reducing the pre-synaptic release of glutamate or interfering with post-synaptic NMDAR-regulated signaling pathways. The efficacy of such control requires the endocannabinoid system to apply its negative influence in a manner that is proportional to the strength of NMDAR signaling. Thus, cannabinoids acting at the wrong time or exerting an inappropriate influence on their receptors may cause NMDAR hypofunction. The purpose of the present review is to draw the attention of the reader to the newly described functional and physical CB1-NMDAR association, which may elucidate the scenario required for the rapid and efficacious control of NMDAR activity. Whether alterations in these mechanisms may increase NMDAR hypofunction leading to vulnerability to schizophrenia will be outlined.

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[90]
Howes O,McCutcheon R,Stone J. Glutamate and dopamine in schizophrenia:an update for the 21st century[J].J Psy-chopharmacol,2015,29(2):97-115.doi:10.1177/0269881114563634.

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