人体生理学 (8).pdf

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1、,*FacultyofMedicine,MolecularandClinicalPharmacology,ICBM,University ofChile,Santiago,ChileDepartamento de Ciencias B?asicas,Facultad de Ciencias,Universidad Santo Tom?as,ChileInstitute of Biomedical Technologies National Research Council of Italy,Segrate,Milan,ItalyAbstractThe molecular mechanisms

2、causing the loss of dopaminergicneurons containing neuromelanin in the substantia nigra andresponsible for motor symptoms of Parkinsons disease arestill unknown.The discovery of genes associated withParkinsons disease(such as alpha synuclein(SNCA),E3ubiquitin protein ligase(parkin),DJ-1(PARK7),ubiqu

3、itincarboxyl-terminal hydrolase isozyme L1(UCHL-1),serine/threonine-protein kinase(PINK-1),leucine-rich repeat kinase 2(LRRK2),cation-transporting ATPase 13A1(ATP13A),etc.)contributed enormously to basic research towards under-standing the role of these proteins in the sporadic form of thedisease.Ho

4、wever,it is generally accepted by the scientificcommunity that mitochondria dysfunction,alpha synucleinaggregation,dysfunction of protein degradation,oxidativestress and neuroinflammation are involved in neurodegener-ation.Dopamine oxidation seems to be a complex pathway inwhich dopamine o-quinone,a

5、minochrome and 5,6-indolequi-none are formed.However,both dopamine o-quinone and5,6-indolequinone are so unstable that is difficult to study andseparate their roles in the degenerative process occurring inParkinsonsdisease.Dopamineoxidationtodopamineo-quinone,aminochrome and 5,6-indolequinone seems

6、toplay an important role in the neurodegenerative processes ofParkinsons disease as aminochrome induces:(i)mitochon-dria dysfunction,(ii)formation and stabilization of neurotoxicprotofibrils of alpha synuclein,(iii)protein degradation dys-function of both proteasomal and lysosomal systems and(iv)oxi

7、dative stress.The neurotoxic effects of aminochrome indopaminergic neurons can be inhibited by:(i)preventingdopamine oxidation of the transporter that takes up dopamineinto monoaminergic vesicles with low pH and dopamineoxidativedeaminationcatalyzedbymonoaminooxidase(ii)dopamine o-quinone,aminochrom

8、e and 5,6-indolequinonepolymerization to neuromelanin and(iii)two-electron reductionof aminochrome catalyzed by DT-diaphorase.Furthermore,dopamine conversion to NM seems to have a dual role,protective and toxic,depending mostly on the cellular context.Keywords:5,6-indolequinone,aminochrome,dopamine,

9、dopa-mine o-quinone,neurodegeneration,neuromelanin,Parkinson0sdisease.J.Neurochem.(2014)129,898915.Dopamine metabolismDopamine synthesis and storageDopaminergic neurons are involved in motor activity,inwhich dopamine is synthesized,stored and released tointersynaptic space.Dopamine is synthesized fr

10、om the aminoacid tyrosine in two steps that occur in the cytosol:(i)hydroxylationoftyrosinetol-dihydroxyphenylanaline(l-dopa),a reaction catalyzed by tyrosine hydroxylase(TH)that requires oxygen and(ii)decarboxylation of l-dopa todopamine,a reaction catalyzed by aromatic amino aciddecarboxylase(AADC

11、)that generates CO2.Interestingly,dopamine synthesis does not result in dopamine accumulationin the cytosol as a consequence that TH and AADC areassociated with the vesicular monoaminergic transporter-2(VMAT-2)generating a complex where tyrosine is convertedto l-dopa that immediately decarboxylated

12、to dopamine.TheReceived February 10,2014;accepted February 12,2014.Address correspondence and reprint requests to Juan Segura-Aguilar,Faculty of Medicine,Molecular and Clinical Pharmacology,ICBM,Santiago,Chile.E-mail:jseguramed.uchile.clAbbreviations used:AADC,aromatic amino acid decarboxylase;COMT,

13、catechol ortho-methyltransferase;GSTM2,glutathione S-trans-ferase M2-2;l-dopa,l-dihydroxyphenylanaline;MAO,monoamineoxidases;NM,neuromelanin;TH,tyrosine hydroxylase;VMAT-2,vesicular monoaminergic transporter-2.898 2014 International Society for Neurochemistry,J.Neurochem.(2014)129,898-915JOURNAL OF

14、NEUROCHEMISTRY|2014|129|898915doi:10.1111/jnc.12686latter is taken up into the monoaminergic synaptic vesicles byVMAT-2(Cartier et al.2010),a dopamine transporterlocalized in the membranes of these vesicles(Miller et al.1999).This complex(TH-AADC-VMAT-2)seems to play animportant role in the preventi

15、on of dopamine oxidation as theprotons of the hydroxyl groups are dissociated when dopa-mine is found in the cytosol at pH 7.4(Linert et al.1996).Therefore,dopamine uptake into monoaminergic vesiclesprevents both the accumulation of free dopamine in thecytosol and the oxidation of dopamine to o-quin

16、one becausethe pH inside monoaminergic synaptic vesicles is 2.02.4 pHunits lower than the pH in the cytosol(Guillot and Miller,2009).At this low pH,the protons of dopamine hydroxylgroups are strongly bound to the oxygen of hydroxyl groups.The low pH of monoaminergic vesicles is because of aVMAT-2-co

17、upled vesicular ATPase,which hydrolyzes ATPto ADP,inorganic phosphate and one proton(H+),creating aproton gradient(Fig.1).The dopamine stored in monoaminergic vesicles isreleased to intersynaptic space to interact with dopaminereceptors in postsynaptic neurons.The clearance of dopa-mine from the syn

18、aptic clefts is mediated by a dopaminetransporter that is localized in the plasma membrane of thedopaminergic neurons.Knockout animals for dopaminetransporter present a dopamine deficiency and decreasedvesicular storage of dopamine(Eriksen et al.2010;Giroset al.1996;Jones et al.1998).The reuptake of

19、 dopaminefrom the synaptic clefts mediated by dopamine transporter isalso a source of free cytosolic dopamine that can be stored inmonoaminergic vesicles by the action of VMAT-2(Fig.1).Dopamine degradation by monoamine oxidaseDopamine excess in the cytosol is degraded by the action ofthe enzyme mono

20、amine oxidase(MAO,E.C.1.4.3.4)whichcatalyzes the oxidative deamination of the dopamine aminogroup to 3,4-dihydroxyphenylacetaldehyde with concomitantformation of ammonia andhydrogen peroxide.The product ofthis reaction is metabolized by aldehyde dehydrogenase to3,4-dihydroxyphenylacetic acid using N

21、AD as electron dona-tor(Fig.2).The MAO enzymes are flavoenzymes containingFAD(Bach et al.1988)where the isozymes labeled as A andB share 70%of primary structure identity.MAO enzymes arefound at the outer membranes of mitochondria in neurons andglia cells(Weyler et al.1990;Shih et al.1997).MAO-B isex

22、pressedinserotonergicandhistaminergicneuronsaswellasin astrocytes(Westlund et al.1988;Saura et al.1994)whereas monoamine oxidase type A(MAO-A)is primarilyexpressed in catecholaminergic neurons.MAO-A has higher affinity for serotonin,dopamine,norepinephrine and epinephrine,whereas the substrates ofMA

23、O-B with high affinity include tyramine,phenylethyl-amine and MPTP(Shih 1991;Strolin-Benedetti et al.1992;Cases et al.1995).However,MAO-B also uses as substratesdopamine,serotonin and norepinephrine(Geha et al.2002).Fig.1 Dopamine synthesis.In the presence of oxygen tyrosinehydroxylase(TH)catalyzes

24、the conversion of the amino acid tyrosineto l-dopa that it is substrate for aromatic amino acid decarboxylase(AADC)that catalyzes the formation of dopamine and CO2.TH andAADC enzymes form a kind of complex with the vesicular monoam-inergic transporter-2(VMAT-2)that is localized in the membrane ofmon

25、oaminergic vesicles.The complex of TH-ADDC-VMAT-2 preventsthe release of dopamine into the cytosol since the formed dopamine isdirectly transported into monoaminergic vesicles.The monoaminergicvesicles have an ATPase that pump up protons into the vesicles usingATP,generating a pH decrease and proton

26、 gradient that is coupled todopamine uptake mediated by VMAT-2 because for one dopaminemolecule uptake into the vesicles one proton is released to the cytosol.The low pH inside the monoaminergic vesicles is essential to storehigh concentration of protonated dopamine hydroxyl groups.2014 Internationa

27、l Society for Neurochemistry,J.Neurochem.(2014)129,898-915Dopamine oxidation and Parkinson0s disease899MAO-A plays a role in maintenance of low concentrations ofdopamine in the cytosol and it has been suggested that itplays a role in oxidative stress because the enzyme generateshydrogen peroxide and

28、 that it is a precursor of hydroxylradicals.Rasagiline has been suggested to protect the brain inParkinsons disease model systems by preventing theformation of reactive oxygen species catalyzed by MAO-B(Weinreb et al.2011).Dopamine degradation by catecholortho-methyltransferaseCatechol ortho-methylt

29、ransferase(COMT;EC 2.1.1.6)alsoparticipates in dopamine degradation by catalyzing themethylation of dopamine to 3-methoxytyramine.Interest-ingly,MAO also catalyzes the oxidative deamination of 3-methoxytyramine to 3-methoxy-4-hydroxyphenylacetalde-hyde.Another enzyme participating in dopamine degrad

30、ationis aldehyde dehydrogenase that catalyzes the oxidation of 3-methoxy-4-hydroxyphenylacetaldehyde to homovanillic acidwith concomitant formation of NADH.However,COMTalso catalyzes the formation of homovanillic acid bymethylating the metabolite formed during MAO-dependentdopamine degradation(see F

31、ig.2).Both soluble(S-COMT)and membrane-bound(MB-COMT)isoforms have been reported in microglial andastroglial cells.COMT is also found in pyramidal neurons,cerebellar Purkinje,granular cells and striatal spiny neurons(My oh anen et al.2010).MB-COMT is localized to the cellbody,axons and dendrites of

32、rat cortical neurons.The catalytic domain of COMT is localized in the C-terminal that is found in the extracellular space(Chen et al.2011).Inhibitors of COMT such as entacapone have beenused in combination with l-dopa to prolong its half-life(Marin and Obeso 2010).Dopamine oxidation to ortho-quinone

33、sDopamine oxidation to dopamineo-quinone and itscyclization to aminochromeIt has been reported that dopamine is able to oxidize to o-quinones spontaneously in the absence of metal-ion catalystsunder aerobic conditions(Linert et al.1996).One electronoxidation of dopamine catalyzed by oxygen will gene

34、ratedopamine o-semiquinone radical and a molecule of super-oxide(Fig.3,reaction 1).The formed dopamine o-semiq-uinoneradicalhastwopossibilitiestoreact:(i)todisproportionate with a second dopamine o-semiquinoneradical generating one molecule of dopamine o-quinone andone molecule of dopamine(Fig.3,rea

35、ction 2);or(ii)to beone-electron oxidized to dopamine o-quinone by reducingoxygen to superoxide(Fig.3,reaction 3).Several metal ionsare able to catalyze dopamine oxidation such as manganese(III)both in aerobic and anaerobic conditions(Segura-Aguilar and Lind 1989);copper(II)(Paris et al.2001);iron(I

36、II)(Paris et al.2005a,b).Dopamine oxidation can also becatalyzed by enzymes such as xanthine oxidase,cytochromeP450,prostaglandinHsynthase,lactoperoxidaseandFig.2 Dopamine degradation catalyzed bymonoamine oxidase and catechol methylortho-transferase.2014 International Society for Neurochemistry,J.N

37、eurochem.(2014)129,898-915900J.Segura-Aguilaret al.dopamine b-monooxygenase that use a peroxidase activity(Graham et al.1978;Hastings 1995;Segura-Aguilar 1996;Foppoli et al.1997;Galzigna et al.2000;Thompson et al.2000).Interestingly,the enzyme tyrosinase that plays animportant role in the formation

38、of pigments in the skincatalyzes the two-electron oxidation of dopamine to dopa-mine o-quinone(Fig.3,reaction 4)without formation ofdopamine o-semiquinone radical(Jimenez et al.1984;Segura-Aguilar et al.1998).Dopamine o-quinone is notstable at physiological pH(Segura-Aguilar and Lind 1989;Bisaglia e

39、t al.2010)as the amino group of dopaminespontaneously rearranges and undergoes cyclization togenerate leucoaminochrome(Fig.3,reaction 5),whichundergoes oxidation to form aminochrome(Fig.3,reaction6).Dopamine o-quinone has a yellow colour with anabsorption maximum at 390 nm that can only be observedw

40、hen dopamine oxidation is conducted below pH 2.0(Segura-AguilarandLind1989;Hawleyet al.1967;Harrison et al.1968;Bisaglia et al.2010).Dopamine o-quinone has been reported to form adducts with severalproteins such as parkin(LaVoie et al.2005),TH(Xu et al.1998),mitochondrial glutathione peroxidase 4(Ha

41、user et al.2013);dopaminetransporter(Whiteheadet al.2001);quinoprotein adducts(Wang et al.2011),mitochondrialcomplex I,III and V,isocitrate dehydrogenase,superoxidedismutase 2,JC-1,UCHL-1(Van Laar et al.2008,2009);tryptophan hydroxylase(Kuhn and Arthur 1998).Dopamineo-quinone is a transient metaboli

42、te formed during dopamineoxidation to aminochrome at physiological pH and therefore,the question is whether the reports with dopamine oxidationcan be assigned to dopamine o-quinone or the real respon-sible for this effect is aminochrome.The rate constant fordopamine o-quinone cyclization to aminochr

43、ome is only0.15/s whereas the rate of aminochrome rearrangement to5,6-dihydroxyindole is much slower(0.06/min)resulting inaminochrome accumulation(Tse et al.1976;Bisaglia et al.2010).The one dimensional NMR spectra of dopamineoxidation with NaIO4revealed the presence of dopamine,aminochromeand5,6-di

44、hydroxyindolebutdopamineo-quinone was not observed because of the intramolecularcyclization of dopamine o-quinone is too rapid(Bisagliaet al.2007).The question is which is the quinone speciesformed during dopamine oxidation that is relevant for theformation of protein adduct with alpha synuclein,par

45、kin,mitochondrial glutathione peroxidase,dopamine transporter,mitochondria complex I,III and V and other proteins relatedto Parkinson0s disease.The nucleophilic addition of gluta-thione to dopamine o-quinone is more rapid than dopamineo-quinone intramolecular cyclization because the constantrate of

46、the nucleophilic addition of glutathione to dopamineo-quinone was estimated to be 200/s(Tse et al.1976).Glutathione S-transferase M2(GSTM2)catalyzed glutathi-one conjugation of dopamine o-quinone to 5-S-glutathionyl-dopamine the structure of which was determined by NMR(d6.77 and 6.87 ppm)and exhibit

47、ed two peaks at 257 and292 nm(Dagnino-Subiabre et al.2000).The non-enzymaticFig.3 Dopamineoxidationtoamino-chrome.Dopamine at physiological pH isable to oxidize to aminochrome in thepresence of oxygen.Reducing oxygen tosuperoxide radical,dopamine can be one-electronoxidizedtodopamineo-semiquinonerad

48、ical(reaction1),whichisabletodisportionatewithanotherdopamineo-semiquinone radical and to generate onemolecule of dopamine o-quinone and onemoleculeofdopamine(reaction2).Alternatively,dopamineo-semiquinoneradical can be one-electron oxidized todopamine o-quinone by reducing oxygen tosuperoxide radic

49、al(reaction 3).Dopaminecanalsobetwo-electronoxidizedtodopamine o-quinone,a reaction that it iscatalyzedbytyrosinase(reaction4).Dopamine o-quinone at physiological pHspontaneouslycyclizestoleucoamino-chrome that autoxidizes to form amino-chrome,reducing oxygen to superoxideradical.2014 International

50、Society for Neurochemistry,J.Neurochem.(2014)129,898-915Dopamine oxidation and Parkinson0s disease901formation of 5-S-glutathionyldopamine was also determinedby NMR when dopamine was oxidized in the presence ofglutathione(Bisaglia et al.2010).On the other side amino-chrome conjugation with glutathio