1、2013諾貝爾生理學(xué)與醫(yī)學(xué)獎授予3名美德科學(xué)家 2013年10月07日 2013諾貝爾生理學(xué)或醫(yī)學(xué)獎 授予 James E. Rothman, Randy W. Schekman （美國）& Thomas C. Sdhof （德國），以表彰他們在細(xì)胞的囊泡運(yùn)輸機(jī)制研究中的杰出貢獻(xiàn)。神經(jīng)元之間的信息傳遞突觸傳遞（Synaptic transmission)突觸（Synapse) 電突觸 (electric synapse) (少見） 化學(xué)突觸 (chemical synapse) （主要形式） - through 神經(jīng)遞質(zhì)（neurotransmitter) Acetylcholine Glut

2、amate GABA Serotonin Noradrenaline Adrenaline Dopamine Histamine 神經(jīng)遞質(zhì) 特定的化學(xué)物質(zhì) 負(fù)責(zé)突觸處信號的化學(xué) 傳遞Monoamines (單胺類遞質(zhì))兒茶酚胺 (catecholamine,CA)多巴胺（Dopamine, DA)去甲腎上腺素（ Noradrenaline, norepinephrine, NA或NE)腎上腺素（ adrenaline, epinephrine, A或E)吲哚胺 (indolamine)5-羥色胺（ Serotonine, 5-HT)兒茶酚Serotonin Pathways in CNS Ne

3、urochemistry Receptors Central FunctionsContents1.1 PATHWAYS in CNS cell body location projection pathway1962年， Falk-Hillarp technique(甲醛誘發(fā)熒光法)Formaldehyde vapourmonoaminebright-green flurorescence (DA, NE) yellow flurorescence (5-HT)1972年瑞典學(xué)者Bjorklund和Lindvail創(chuàng)立了乙醛酸誘發(fā)熒光法單胺類神經(jīng)遞質(zhì)腦內(nèi)分布的觀察手段2. Use of im

4、munofluorescence technique Enzymes，transporters，1. Use of fluorescence histochemistry (Falck & Hillarp)單胺類神經(jīng)遞質(zhì)腦內(nèi)分布的觀察手段 Major Noradrenergic (blue)去甲腎上腺素, Dopaminergic (red)多巴胺 and Serotonergic (green) 5-羥色胺 Pathways in the CNS A8 (lateral, caudal, dorsal to A9) A9 (substantia nigra, SN 黑質(zhì)） A10 (vent

5、ral to A9, ventral tegmentum,腹側(cè)被 蓋區(qū) ) A12 (arcuate nucleus，弓狀核)A8-A10 contains 70% of the DA neurons in the brain Location of DA cell bodies（多巴胺 胞體分布）A8 A10: midbrain 中腦A11A17: diencephalon 間腦A9A10nigrostriatal pathway:黑質(zhì)-紋狀體通路 （運(yùn)動控制）A8, A9- striatum（紋狀體) caudate nucleus（尾核）, putamen（殼核）, amygdala（杏

6、仁核）mesolimbocortical system:中腦-邊緣皮質(zhì)投射 （情感和認(rèn)知）A10-nucleus accumbent（伏隔核）, olfactory tubercle（嗅結(jié)節(jié)）, prefrontal（前額葉皮層） and perirhinal cortex（嗅皮層）A9,A10-anterior cingulate cortex(前扣帶皮層）tuberoinfundibular system:結(jié)節(jié)漏斗系統(tǒng)（DA能短投射） A12-pituitary gland（垂體） （抑制垂體分泌激素）下行束脊髓(Spinal cord)（調(diào)節(jié)軀體和內(nèi)臟感覺運(yùn)動） projection p

7、athways of DA neurons （神經(jīng)投射通路）nigrostriatal pathway:黑質(zhì)-紋狀體通路 （運(yùn)動控制）A8, A9- striatum（紋狀體) caudate nucleus（尾核）, putamen（殼核）, amygdala（杏仁核）mesolimbocortical system:中腦-邊緣皮質(zhì)投射 （情感和認(rèn)知）A10-nucleus accumbent（伏隔核）, olfactory tubercle（嗅結(jié)節(jié)）, prefrontal（前額葉皮層） and perirhinal cortex（嗅皮層）A9,A10-anterior cingulate

8、 cortex(前扣帶皮層）tuberoinfundibular system:結(jié)節(jié)漏斗系統(tǒng)（DA能短投射） A12-pituitary gland（垂體） （抑制垂體分泌激素）下行束脊髓(Spinal cord)（調(diào)節(jié)軀體和內(nèi)臟感覺運(yùn)動） projection pathways of DA neurons （神經(jīng)投射通路）nigrostriatal pathway:黑質(zhì)-紋狀體通路 （運(yùn)動控制）A8, A9- striatum（紋狀體) caudate nucleus（尾核）, putamen（殼核）, amygdala（杏仁核）mesolimbocortical system:中腦-邊緣皮

9、質(zhì)投射 （情感和認(rèn)知）A10-nucleus accumbent（伏隔核）, olfactory tubercle（嗅結(jié)節(jié)）, prefrontal（前額葉皮層） and perirhinal cortex（嗅皮層）A9,A10-anterior cingulate cortex(前扣帶皮層）tuberoinfundibular system:結(jié)節(jié)漏斗系統(tǒng)（DA能短投射） A12-pituitary gland（垂體） （抑制垂體分泌激素）下行束脊髓(Spinal cord)（調(diào)節(jié)軀體和內(nèi)臟感覺運(yùn)動） projection pathways of DA neurons （神經(jīng)投射通路）nigr

10、ostriatal pathway:黑質(zhì)-紋狀體通路 （運(yùn)動控制）A8, A9- striatum（紋狀體) caudate nucleus（尾核）, putamen（殼核）, amygdala（杏仁核）mesolimbocortical system:中腦-邊緣皮質(zhì)投射 （情感和認(rèn)知）A10-nucleus accumbent（伏隔核）, olfactory tubercle（嗅結(jié)節(jié)）, prefrontal（前額葉皮層） and perirhinal cortex（嗅皮層）A9,A10-anterior cingulate cortex(前扣帶皮層）tuberoinfundibular s

11、ystem:結(jié)節(jié)漏斗系統(tǒng)（DA能短投射） A12-pituitary gland（垂體） （抑制垂體分泌激素）下行束脊髓(Spinal cord)（調(diào)節(jié)軀體和內(nèi)臟感覺運(yùn)動） projection pathways of DA neurons （神經(jīng)投射通路） locus coeruleus (LC 藍(lán)斑) complex -A4,A6 lateral tegmental nuclei 外側(cè)被蓋核 -A1,A3,A5,A7,A2 Location of NA cell bodies（去甲腎上腺素能神經(jīng)元-胞體分布）A1 A7: Pons、medulla 腦橋、延髓LC Projection pa

12、thways of NE neuronsThe locus coeruleus complexLateral tegmental nucleiCerebral cortex HippocampusBrainstem nuclei (sensory)Spinal cordCerebellum ThalamusHypothalamusAmygdala SeptumBrainstem nuclei (motor)Hypothalamus (all nuclei)Frontal cortexOlfactory bulbparaventricular nucleus suprachiasmatic nu

13、cleus藍(lán)斑復(fù)合體視交叉上核Noradrenaline pathways in rat brain Synthesis Storage Release Eliminate Reuptake Metabolism1.2 NEUROCHEMISTRYDA/NEDopamineVesiclesynthesisstoragereleasereceptoreliminationreuptakemetabolism神經(jīng)遞質(zhì)的Neurochemistry 示意圖Enzymes: tyrosine hydroxylase(TH, 酪氨酸羥化酶）Dopa decarboxylase(DDC, 多巴脫羧酶）SY

14、NTHESIS (合成）DA and NA synthesis are SimilarPrecursor: tyrosine(酪氨酸）Synthesis for DA in cytoplasm of CA neurons （兒茶酚胺能神經(jīng)元） Synthesis for NA in vesicles of NA neurons（NA 神經(jīng)元囊泡）Dopamine-hydroxylase (DH) SYNTHESIS NA DAPrecursor:Tyrosine (tyr) tyrEnzymes: tyrosine hydroxylase (TH)* TH*Dopa decarboxylase

15、 (DDC) DDCDopamine-hydroxylase (DH)Cytosol and vesicle cytosolNAergic neurons DAergic SYNTHESIS* rate-limiting enzyme SYNTHESISThe enzymes involved:Tyrosine hydroxylase (TH, 酪氨酸羥化酶）Dopa decarboxylase (DDC, 多巴脫羧酶）Dopamine-hydroxylase (DH,多巴胺羥化酶) SYNTHESISTyrosine hydroxylase (TH, 酪氨酸羥化酶）Cytoplasmic e

16、nzyme： tyrL-dopa Km=510-6 M 腦內(nèi)色氨酸Fe2+,O2, and tetrahydropterine (BH4)Substrate-specificInhibitors: desferrioxamine(去鐵草酰胺); dopamine; PCPATrp: rate-limiting factor!TrpbloodBBBbrainLNAA carriers: large neutral amino acids carriers （大分子中性氨基酸轉(zhuǎn)運(yùn)體：for Trp, Phe, Leu, Ile, Val 轉(zhuǎn)運(yùn)）TrpTPH正常情況下，腦內(nèi)TPH遠(yuǎn)未飽和Neuroc

17、hemistry of Serotonindietary availabilityFM&Q Fig 9.4FM&Q Fig 9.5high carbo (碳水化合物） and low protein diet is best for increasing 5-HT. SYNTHESIS5-HTP decarboxylase (5-羥色氨酸脫羧酶)soluble enzyme： 5-hydroxytryptophan 5-HTKm=10 MSubstrate-specificL-aromatic amino acid decarboxylase (AADC) the same enzyme in

18、volved in the biosynthesis of catecholamines SYNTHESISControl of 5-HT synthesis1, brain Trp level- rate-limiting factor2, brain 5-HT level- negative feedback3, TPH （色氨酸羥化酶） rapid: activity slow: expressionEvidence:Neuronal depolarization phosphorylation:Neuronal depolarization TPH activation Ca2+/ca

19、lmodulin-dependent protein kinase inhibitorPhosphorylation of TPH -an increase in activity Ca2+/calmodulin-dependent protein kinase II cAMP-dependent protein kinase (PKA) SYNTHESIS2.2 STORAGE (儲存） Vesicular storage (30-35 mm diameter) （囊泡儲存） Vesicles containing serotonin-binding proteins (SBP), form

20、 a macromolecular complex with 5-HT SBP can be secreted into the synapse along with 5-HT VMAT （囊泡單胺轉(zhuǎn)運(yùn)體）： 質(zhì)子泵產(chǎn)生的 H+ 梯度作為能量 STORAGE2.3 RELEASE （釋放） 胞裂外排 （exocytosis) Autoreceptors suppress 5-HT release: 5-HT1A receptors: on the cell bodies of neurons in the Raphe nuclei ; 5-HT1B/1D receptors: on their

21、 terminals Heteroceptors modify 5-HT release: nAChR: increase release from striatal synaptosomes; 2A adrenoceptors: depress cortical release Releasing agent: fenfluramine2.4 REUPTAKE （重攝?。﹕erotonin transporter (SERT) Na+/Cl- dependent transporterSERT has large number of potential phosphorylation sit

22、es and can be phosphorylated by PKA and PKC. In vitro SERT phosphorylation by PKC leads to the internalization of the protein and to a reduction in serotonin reuptake.“uptake-1” and “uptake-2”.FM&Q Fig 9.1Dependent on Na+ and Cl- bindingNa+ and Cl- are brought into the cell along with 5-HT and K+ is

23、 extrudedDrugs that block 5-HT reuptake bind to the 5-HT binding siteEnergy supplied by Na+/K+ dependent ATPasePhosphorylation of SERTAt least 8 phosphorylation siteRapid, short-term regulationPKA, PKC: internalization (decrease the reuptake of 5-HT)Inhibitors of 5-HT transporter (SERT) TCA（Tricycli

24、c antidepressants，三環(huán)類抗抑郁藥）: clomipramine(氯丙咪嗪)， Selective serotonin reuptake inhibitor (SSRIs): fluoxetine, sertraline, fluvoxamine, paroxetine, citalopram Cocaine （可卡因） Amphetamine （安非他命） Fenfluramine（芬弗拉明）(DAT, SERT) MDMA（ecstasy，搖頭丸）：neurotoxin for 5-HT neuron(similar to the effect of MPTP on DA

25、neuron) （SERT底物，刺激腦部快速釋放大量的5-HT）2.5 METABOLISM （酶解失活）MAOA- CHO- COOH5-HT 5-HIAA（5-羥吲哚乙酸）醛脫氫酶MOAI（-）NCNCNH2COOHCOOHNH2OHNCNH2OHHTryptophan5-Hydroxytryptophan5-HydroxytryptamineNCCOOH5-OH Indole Acetaldehyde5-Hydroxy Indole Acetic AcidTryptophan hydroxylase5-OH Tryptophan decarboxylaseMAOAldehyde dehy

26、drogenase(Rate limiting)In diet. ActiveCNS transport 7 distinct families 14 distinct subtypes have been cloned so far3. RECEPTORS（5-HT受體）The pharmacology of 5-HT is extremely complex, with its actions being mediated by a large and diverse range of 5-HT receptors - many of which have poorly character

27、ised physiological functions.All are G protein coupled receptors except 5-HT3 5-HT3 : ligand-gated ion channels: 5-HT3A， 5-HT3B， 5-HT3CSplice variants: 5-HT3, 5-HT4 , 5-ht6, 5-HT7 RNA edited isoforms: 5-HT2C Different central distribution and function5HT1A: role in anxiety/depression5HT1D: role in m

28、igraine5HT2: role in CNS various behaviors, and in cardiovascular system5-HT3: role in nausea and vomiting esp. due to Chemotherapy.5-HT1A: hippocampus, septum, amygdala, dorsal raphe, cortex5-HT1B: substantia nigra, basal ganglia5-HT1D: substantia nigra, striatum, accumbens, hippocampus less than 5

29、-HT1B5-ht1E, 5-ht1F： lack of specific ligands 5-HT1A receptors: somatodendritic autoreceptors reside on serotonergic cell bodies and dendrites reduce cell firing and curtails the synthesis and release of 5-HT 5-HT1B/1D receptors: reside on presynaptic nerve terminal decrease the local synthesis and

30、release of 5-HTAutoreceptors on somatodendrites and terminals5-HT2A: cortex, olfactory tubercle, claustrum5-HT2B: not located in brain (lack of specific ligands)5-HT2C: basal ganglia, choroid plexus, substantia nigra5-HT3 receptor: Spinal cord, cortex, hippocampus, brain stem nucleia ligand-gated io

31、n channel that is homologous to other receptor ionophores.nonselective cation channel; triggers a rapid, transient depolarizing current that is carried by Na+ and K+.5-HT4: hippocampus, nucleus accumbens, striatum, substantia nigra5-ht5A: cortex, hippocampus, cerebellum5-ht5B: hippocampus5-ht6: stri

32、atum, olfactory tubercle, cortex, hippocampus5-HT7: hypothalamus, thalamus, cortex, suprachiasmatic nucleusSerotonin Agonists （受體激動劑）Sumatriptan(舒馬曲坦): 5-HT1D agonist; antimigraineBuspirone(丁螺環(huán)酮) : 5-HT1A agonist for anxietyCisapride(西沙比利): 5-HT4 agonist to GI motility and decrease GE reflux (Remove

33、d from US market due to fatal arrhythmias)LSD: 5HT1A agonist hallucinogen(致幻劑)Serotonin Antagonists（受體拮抗劑）Methysergide（二甲麥角新堿） and Cyproheptadine（賽庚啶）: 5HT2 antagonists. In carcinoid, migraine. Ketanserin（酮舍林,凱坦生）: 5HT2 and Alpha antagonist used as antihypertensive.Ondansetron（奧坦西?。? 5-HT3 antagonis

34、t for chemotherapy induced nausea and vomitingClozapine（氯氮平）: 5HT2A/2C antagonist: for schizophrenia.Interference with 5-HT systema shift back to less selective compoundsInterference with 5-HT system Levels of serotonin in the brain can be manipulated by:Inhibit tryptophan uptake into CNS: exclusion

35、 of tryptophan from the dietInhibit synthesis: p-chlorophenylalanine (irreversible) （TH）Inhibit neuronal re-uptake: cocaine, SSRI (e.g. fluoxetine), TCA (e.g. imipramine丙咪嗪)Inhibit storage-deplete: reserpineInhibit metabolism: MAO inhibitorsPromote release: p-chloroamphetamine - then depletes (e.g.

36、fenfluramine to appetite)Neurotoxins:5,6-DHT/5,7-DHTNon-selectiveAlthough serotonin can mediate numerous therapeutic actions, too much serotonin can cause dangerous toxicity. The blind men and the elephant. 5-HT, 5-hydroxytryptamine or serotonin;CBF, cerebral blood flow; GI, gastrointestinal. Figure

37、 adapted from a woodblock print by Katsushika Hokusai (17601849).4. CENTRAL FUNCTIONS Pain perception （projection from inferior group neurons to spinal cord) Sleep/Wakefulness Various behaviors normal/abnormal: depression, schizophrenia, obsessive compulsive behavior, etc. DepressionOld Man with his

38、 Head in his HandsVan Gogh, 1882 Depression Catecholamine HypothesisFifteen percent of individuals prescribed reserpine for hypertension developed depression.MAOIs being prescribed for tuberculosis (肺結(jié)核） were found to elevate mood.Cocaine and amphetamine, which increase catacholamines, elevate mood.

39、NE is found in abundance in the limbic systems（大腦邊緣系）and is thought to regulate mood.Depressed individuals show low levels of MHPG, a metabolite of NE.Tricyclic antidepressant drugs were found to inhibit the reuptake of NE.(抑郁癥可能與CA的功能不足/降低有關(guān)）Problems with the Catacholamine HypothesisTryicyclic anti

40、depressant drugs required several days or weeks to alleviate the symptoms of depression.Healthy individuals did not show elevated mood with tricyclic antidepressants.Cocaine and amphetamine are not good antidepressants.Non-tricylcic antidepressants that specifically affected 5-HT were found to be cl

41、inically affective in alleviating the symptoms of depression.Depression - Serotonin Hypothesis (抑郁癥可能與5-HT的功能不足/降低有關(guān)）The most clinically effective antidepressants are those that are most selective for reuptake of 5-HT (SSRIs).The data that previously were taken in support of the catacholamine hypoth

42、esis could also support the role of 5-HT because reserpine, MAOIs and tricyclics also affect 5-HT.But the effects of SSRIs also take days or weeks even though the biochemical action on 5-HT reuptake is immediate. Depression - Support for the Serotonin HypothesisDepressed individuals have:Lower level

43、s of plasma tryptophan.Lower CSF levels of 5HIAA.Decreased platelet （血小板）5-HT uptake suggesting lower levels of 5-HT release.Blunted responsiveness to serotonin agonist challenge suggesting decreased 5-HT responsiveness.Specific Treatments for DepressionAll effective antidepressants act by increasin

44、g the activity of one or both of these neurotransmitter systemsBlock neurotransmitter reuptake systemsSerotonin Re-uptake inhibitors (SSRIs) Tricyclic antidepressants (TCAs)Block neuronal metabolism and so increase amount releasedMonoamine Oxidase Inhibitors (MAOIs)Increase amount of neurotransmitte

45、r releasedPresynaptic autoreceptor blockadeElectroconvulsive Therapy (ECT)More effective than drug therapiesRapid therapeutic effect (1-2 weeks vs 4-6 weeks for other therapies)Minimal adverse effectsPossible Sites of Antidepressant Drug Action Monoamine Oxidase InhibitorsAutoreceptor AntagonistsMir

46、tazepineRe-Uptake InhibitorsSSRIsTCAs Requirments1）Pathways and distribution of DA,NA and 5-HT in CNS (Substantia nigra, locus coeruleus, or raphe nucleus)2）Neurochemistry of DA,NA and 5-HT synthesis（enzymes and regulation factors: TH, DDC, DbH; TPH, 5-HTPDC; Rate-limiting enzyme） storage (Vesicle,

47、VMAT; H+ dependent transporter; Reserpine) release reuptake (DAT, NET, SERT, Na+/Cl- dependent transporter; Cocaine, amphetamine, TCAs, SSRIs, MPTP, 6-OHDA) metabolism (MAO, COMT, MAOI )3）Receptors of DA,NA and 5-HT Classification (D1D5; a1, a2, b; 5HT1.5HT7 receptors; autoreceptors) mechanisms (ago

48、nist, antagonist)4） Central function DA & Parkinsons disease (PD) DA & Schizophrenia Monoamines (NA & 5-HT) & Depression 謝 謝！ Roberto Maggio1 and Mark J Millan, Current Opinion in Pharmacology 2010, 10:100107Homomers: D2D2 Heteromers: D2D3, D1/D5, D2/D3/D4, 磷酸化的DARPP-32是蛋白磷酸酯酶-I (PP1)的強(qiáng)大抑制劑，阻止PP1的去磷

49、酸化作用，使生理效應(yīng)增強(qiáng)。其重要生理意義：1，DARPP-32達(dá)到最大半數(shù)抑制效應(yīng)時，所需的濃度極低（10nmol/L），而在神經(jīng)元側(cè)棘中DARPP-32的濃度比上述抑制作用濃度高1000-5000倍；2，其 抑制作用完全依賴于分子結(jié)構(gòu)中Thr 34 的磷酸化，一旦被去磷酸化，抑制作用隨之解除；3，腦內(nèi)只有磷酸酯酶-I受到磷酸化的DARPP-32的抑制，故有極好的選擇性，增強(qiáng)D1受體的生理效應(yīng)，放大D1受體的作用。4,磷酸化的DARPP-32本身被蛋白磷酸酯酶2B去磷酸化后，則失去抑制磷酸酯酶I的作用。Regulation of DARPP-32 phosphorylation by four

50、kinases and three phosphatasesNeuropharmacology 47 (2004) 1423；Cell. Mol. Life Sci. 65 (2008) 2125 2127學(xué)生課后提問-2012-10 ：華山醫(yī)院神經(jīng)內(nèi)科一學(xué)生：他們擬用影像學(xué)方法標(biāo)記和檢測人腦黑質(zhì)（LC）中的VMAT，用于分析LC腦區(qū)DA neuron 在不同病程階段 PD病人的特征。 此時，黑質(zhì)中的 NA，或5-HT 神經(jīng)元（其中同樣也包含 VMAT ）是否會對其分析產(chǎn)生影響？學(xué)生課后提問-2011-10 ：已知PKC對 TH的 Ser 40 磷酸化有促進(jìn)作用，并增強(qiáng)其活性。 但在隨后的論文介紹中， PKC-delta 卻是通過增強(qiáng)PP2B 的磷酸化和活性，從而抑制TH 活性的。 那么， PKC-delta 究竟是否也能通過直接磷酸化TH的Ser40 從而增強(qiáng)其活性，只是該途徑的效應(yīng)要小于 PP2B 途徑 ？（從而使總的效應(yīng)是： PKC-delta 降低TH 磷