Proc. Nati. Acad. Sci. USAVol. 91, pp. 7772-7776, August 1994Neurobiology

High levels of a retinoic acid-generating dehydrogenase in themeso-telencephalic dopamine system

(bas gania/corpus siatum/nucleus accumbens/disfram/Parkluson die)

PETER MCCAFFERY AND URSULA C. DRAGERDivision of Developmental Neuroscience, E. K. Shriver Center, Waltham, MA 02254; and Department of Psychiatry, Harvard Medical School,Boston, MA 02115

Communicated by Ann M. Graybiel,* April 25, 1994 (received for review January 15, 1994)

ABSTRACT Retinoic acid is synthesized from retinalde-hyde by several different dehydrogenases, which are arrangedin conserved spatial and developmentally regulated patterns.Here we show for the mouse that a class-i aldehyde dehydro-genase, characterized by oxidation and disulfiram sensitivity,is found in the brain at high levels only In the basal forebrain.It is present in axons and terminals of a subpopulation ofdopaminergic neurons of the mesostriatal and meimbicsystem, forming a retinoic acid-generating projection from theventral tegmentum to the corpus striatiu and the shell of thenucleus accumbens. In the striatum the projection is heaviestto dorsal and rostral regions, dining gradually towardventral. The enzyme is expressed early in development, shortlyafter appearance of tyrosine hydroxylase. Otherd nergicneurons in the brain, as well as the chromaffin cells of theadrenal medulla, do not contain this dehydrogenase. Thepresence ofthis enzyme may be a factor in the long-term successof transplants of dopaminergic cells to the corpus stratum InParkinson disease, and it may play a role in parkinsonism andcatatonia due to disulfiram (Antabuse) neurotoxicity.

Aldehyde dehydrogenases represent a diverse family of re-lated enzymes involved in the oxidation of exogenous andendogenous aldehydes (1-3). They differ in a range of char-acteristics including substrate selectivity: of 13 isoformsidentified in the mouse, only class 1 (cytosolic) aldehydedehydrogenase was found to be capable of oxidizing retinal-dehyde to retinoic acid (4). Although in vitro this isoform canoxidize a broad range of substrates, retinoic acid productionis considered its main physiological role. In addition to class1 aldehyde dehydrogenases, several recently reported dehy-drogenases are capable of generating retinoic acid (5). Ex-pression of the different retinoic acid-generating dehydroge-nases occurs in stereotypic spatial and developmentallyregulated patterns that are similar in mammals, birds, andcold-blooded vertebrates. For instance, the embryonic retinaof all vertebrates tested contains in its dorsal part a class 1aldehyde dehydrogenase, termed AHD2 in the mouse, and inits ventral part different dehydrogenases (5, 6).

MATERIALS AND METHODSRetinoic Acid Assays. All determinations of retinoic acid

shown here make use of a reporter cell line: teratocarcinomacells transfected with a sensitive retinoic acid-response ele-ment driving (3-galactosidase expression (7). These cellsspecifically detect retinoic acid, the product of the irrevers-ible dehydrogenation reaction of retinaldehyde, but they donot give information on the preceding enzymatic reaction, thereversible oxidation of retinol to retinaldehyde, which iscatalyzed by an alcohol dehydrogenase (8). The cells areexposed to a retinoic acid-containing sample overnight,

fixed, and treated with 5-bromo-4-chloro-3-indolyl f3-D-galactopyranoside (X-Gal), and the calorimetric reaction isquantified in an ELISA reader. The cells are much moreresponsive to all-trans-retinoic acid than the cis isomers, anda doubling in colorimetric readings indicates a 10- to 100-foldincrease in retinoic acid. We did not attempt to measureabsolute retinoic acid levels, but all measurements shownrepresent comparisons of different samples processed inparallel; no values are given for the calorimetric readings, asall information is contained graphically in the comparisons.We did the following three assays based on the reporter cells.Zymography bioassay. This assay (Fig. 1) involves sepa-

ration of tissue homogenates in parallel lanes by isoelectricfocusing, cutting the lanes into thin slices, eluting the proteinsinto L15 tissue culture medium in 96-well plates, and testingfor retinoic acid synthesis from 50 nM retinaldehyde in thepresence of 2 mM NAD (5). This assay probably detects allenzymes capable of generating any form of retinoic acid.Enzyme lability assay. Similar-size tissue pieces were

dissected from the striatum and olfactory bulbs of youngadult mice and were subjected to one of two pretreatmentsbefore being assayed for retinoic acid synthesis from addedretinaldehyde and NAD with the reporter cells (Fig. 2a). (i)The tissues were homogenized in L15 medium without anyadditives or with 2.5 1LM or 12.5 ,uM disulfiram and kept onice, or (ii) the tissues were homogenized with or without 1mM dithiothreitol and kept at 370C for 10 or 20 min; thecontrol here is a 20-min air exposure at 370C in the presenceof dithiothreitol. Like the effect of air exposure, the dis-ulfiram effect can be prevented by dithiothreitol.Estimates of endogenous retinoic acid levels. For this

assay (Fig. 2b), similar-size samples from adult olfactorybulb, striatum, and hippocampus were homogenized in L15medium in the presence of 2 mM NAD, but no retinaldehydewas added. The homogenates were incubated overnight at370C, and the supernatants were plated in serial dilutions ontothe reporter cells.

Histology. All murine tissue, derived from an outbred mousecolony, was fixed in periodate-lysine-paraformaldehyde (9),either by immersion (embryos) or by transcardiac perfusion(adult); the monkey tissue came from an adult rhesus macaqueperfused with 4% paraformaldehyde for an unrelated experi-ment. Cryostat sections were labeled with 2 different antiserato rodent class 1 aldehyde dehydrogenase (10, 11) and with amonoclonal antibody or an antiserum to tyrosine hydroxylase(TH; Boehringer Mannheim; Eugene Tech). Antibody bindingwas visualized with fluorescent secondary antisera.

RESULTSIn the embryonic mouse eye, retinoic acid is generated byfour zymographically distinct enzymatic activities (Fig. la,

Abbreviations: AHD2, murine class 1 (cytosolic) aldehyde dehydro-genase; TH, tyrosine hydroxylase.*Communication of this paper was initiated by W. J. H. Nauta and,after his death (March 24, 1994), completed by Ann M. Graybiel.

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Proc. Natl. Acad. Sci. USA 91 (1994) 7773

a AHD2 Vl V2 V3

u A

toNr.

b

basioctory bulb

hippocampus

basic < > acidicbasic < > acidic

FIG. 1. (a) Zymographs of retinoic acid-generating enzymes insimilar-size samples dissected from the corpus striatum, olfactorybulb, and hippocampus of adult mice, processed in parallel with anembryonic day 16 (E16) eye, which serves here as a pH/enzymemarker; the designations of the eye enzymes are indicated at the top(6). No numerical values are given for the colorimetric readings, asonly the comparative values, depicted graphically, are of signifi-cance. For easier comparison of enzyme activities, the colorimetricreading traces are aligned and offset. The basic enzyme activity isAHD2; the acidic activities in the brain samples represent noveldehydrogenases resembling those in the embryonic eye. (b) Devel-opmental changes in retinoic acid-generating enzymes in the devel-oping striatum. Similar-size samples were dissected from the stria-tum of E16, postnatal day 2 (P2), and P27 mice and were processedin parallel by zymography.

top trace): one basic enzyme representing AHD2 localized indorsal retina, and three acidic activities, V1, V2, and V3,mainly localized in ventral retina (V1 and V3) and the retinalpigment epithelium (V2) (5,6). We screened different regionsof the central nervous system from forebrain to spinal cordfor zymographically detectable retinoic acid-generating de-hydrogenases, starting from embryonic day 8 (E8, with dayof conception = EO.5) to adulthood. In general, levels ofretinoic acid-generating enzymes were much higher in thedeveloping nervous system than in the mature system, and atany given age enzyme levels varied substantially betweendifferent regions-e.g., embryonic-retina and spinal-cordlevels were very high, and brain levels were overall much

ato100%1 111

'~60%! U

20 0 2.5 12.5 0 10' 20' 0 2.5 12.5 0 10' 20'disulfiram 02/370C disulfiram 0 /370C

[AM] [PM] 2

olfactory bulbstriatum

hippocampuscolorimetric readings

FIG. 2. (a) Comparisons of the decay of retinoic acid-generatingenzyme activity in homogenates of the striatum and olfactory bulbexposed to disulfiram or to room air. Note the much more pro-nounced activity decline in the striatum as compared with theolfactory bulb. (b) Endogenous retinoic acid content, or synthesisfrom endogenous precursor, in homogenized, similar-size samplesfrom adult olfactory bulb, striatum and hippocampus.

lower. Acidic dehydrogenases were detectable throughoutthe brain, but their compositions and levels varied: in mostregions in both developing and mature brain, high activitywas associated with the pia. In the adult brain the relativelyhighest activity was present in the olfactory bulbs, whereseveral different enzymes were expressed, and only lowlevels were found in the hippocampus, which containedmainly a single activity (Fig. la, bottom traces). AHD2 wasdetectable in the brain at high levels only in one area: thestriatum (Fig. la). Whereas in the adult striatum all retinoicacid synthesis was mediated by AHD2, the fetal striatumcontained rather high levels of acidic dehydrogenases similarto those in the embryonic eye; the basic AHD2 took overslowly during the perinatal period (Fig. lb).

In in vitro assays with substrates other than retinoic acid,the class 1 isoform is known to stand out from other mem-bers of the family of nonspecific aldehyde dehydrogenasesthrough its very high susceptibility to oxidation and toinhibition by the sulfhydryl reagent disulfiram (12, 13). Thesame distinction applies to a comparison of AHD2 with thenovel retinoic acid-generating dehydrogenases, which prob-ably do not belong to the nonspecific aldehyde dehydroge-nase family (14): enzyme activity in homogenates of adultstriatum was much more susceptible to oxidation and dis-ulfiram treatment than activity in olfactory-bulb homoge-nates (Fig. 2a). Like the effect of air exposure, the disulfirameffect could be prevented and, at least partially, reversed bythe reducing reagent dithiothreitol; it is possible that bothmanipulations target the same sulfhydryl groups on AHD2.The exact location of the enzymes in the fetal striatum is

not clear; they might be associated with the germinal zonegiving rise to the striatum, the ganglionic eminence, whichcontaminated the dissected samples. For AHD2, however,antisera specific for rodent class 1 aldehyde dehydrogenaseallow a more detailed analysis (10, 11). Immunohistochemicalexamination of the adult striatum showed bright AHD2labeling, all localized in axons and synaptic terminals (Fig. 3a and b). Cell bodies of origin were exclusively located in thenigral-ventral tegmental areas A9, A8, and A10. Double-labeling with a monoclonal antibody to TH showed AHD2 ina subpopulation of the dopaminergic neurons: most double-labeled somata were found in the nigral pars compacta (A9;Fig. 4 a and b), fewer in the ventral portions of A8, and onlyan occasional one in A10 (Fig. 4 c and d). The projectionsfrom these neurons, relatively concentrated in rostral andventral portions of the tegmental dopamine system, corre-spond to the known topography of mesostriatal and mesolim-bic projections (15): AHD2-containing fibers were densest inmost rostral and dorsal portions of the striatum, with thedensity gradually declining toward ventral (Fig. 3 a and c). Ofthe mesolimbic projection sites, only the shell of the nucleusaccumbens received heavy AHD2 input (Fig. 3 a and c,arrow). AHD2 projection to the accumbens core and olfac-tory tubercle was much weaker than the dopaminergic input(Fig. 3 a, c, and d), and to the septal area both projectionswere sparse. No AHD2 input to the cortex was found. Otherdopaminergic cells, including the ones in the hypothalamus,olfactory bulb, retina, and adrenals, did not contain AHD2.Zymographically the adrenal gland did contain low levels ofAHD2 (not shown); double-labeled immunohistochemicalpreparations, however, showed no AHD2-like immunoreac-tivity in dopaminergic chromaffin cells, but only in someadjoining cortical and in nondopaminergic medullary cells(Fig. 4 e and f).

In immunohistochemical screens of the developing murinenervous system, from E8 onward, expression of AHD2 indopaminergic neurons was found to begin about 1 day afterexpression of TH (16). In double-labeled sections throughE12 mice, TH-positive cells were present in the mesenceph-alon, but they did not contain AHD2. At E13 double-labeled

Neurobiology: McCaffery and DrAger

striahim nlf^dnr% h..lls

7774 Neurobiology: McCaffery and Drager

FIG. 3. Coronal sections through the striatum of young adult mice double-labeled with antisera to rodent class 1 aldehyde dehydrogenase(10, 11) (a-c) and a monoclonal antibody toTH (d). (a) Low-power view ofone cerebral hemisphere to illustrate the dorsoventrally graded AHD2distribution. In addition to the nigrostriatal system, both class-i antisera label the brain surface/pia; as zymographs show no AHD2 here, thisprobably represents a dehydrogenase that is immunologically similar to AHD2. (b) High-power view ofthe striatum edge showing AHD2-positivefibers ending in bright specks, presumably synaptic terminals. (c and d) Partial view of the striatal-limbic transition, double-labeled for AHD2(c) and TH (d). Note the relatively homogeneous TH distribution; by contrast, the AHD2 input is weak to most of the limbic regions exceptfor heavy input to the shell of the nucleus accumbens (arrow). AHD2 is detected by fluorescein isothiocyanate immunofluorescence, and THis detected by rhodamine isothiocyanate. [Bars = 1 mm (a), 50 inm (b), and 300 pim (c and d).]

cells were found, and a few AHD2-positive fibers couldalready be detected in the beginning medial forebrain bundle(Fig. 5a, arrows); by E14 this projection was quite pro-nounced (Fig. Sb, arrows). The dopaminergic cell bodies oforigin in the mesencephalon were very brightly labeled withthe AHD2 antiserum, as illustrated for E16 (Fig. Sc). Al-though at E16 the AHD2 projection to the striatum was quiteobvious immunohistochemically (not shown), it was not yetdetected by the less-sensitive zymography assay (Fig. lb).

Preliminary examinations of sections through the substantianigra from an adult monkey (Macaca fasciculars), double-labeled with the same antibodies, revealed a similar selectiveclass 1 aldehyde dehydrogenase labeling ofa subpopulation ofdopaminergic neurons, with double-labeled cells concentratedin ventral regions (not shown). This makes it likely that thepattern described here for the mouse extends to primates.

Although retinoic acid synthesis is considered the mainrole of class 1 aldehyde dehydrogenase, it was necessary to

demonstrate such afunction in the striatum. A sensitive assayfor endogenous retinoic acid synthesis in small samples isovernight coculture of test tissues in tight contact with theretinoic acid reporter cells (7). Because it was not possible todissect the few AHD2-containing cell bodies clean enoughfrom other tegmental cells to assure good contact and be-cause overnight culture of disrupted terminals embeddedbetween live cells in the striatum was problematic, thereporter cells were used to assay homogenized tissue. Noretinaldehyde is added in this assay, and all detected activityreflects either endogenous retinoic acid content or synthesisfrom endogenous retinoid precursors. In embryonic tissues,assays ofhomogenates correlate well with tissue explants (5).Fig. 2b shows such comparative retinoic acid estimates forsimilar-size samples from striatum, olfactory bulbs, andhippocampus. Consistent with zymographically detectableenzyme activities (Fig. la), retinoic acid levels were very lowin the hippocampus and considerably higher in both striatum

Proc. Natl. Acad Sci. USA 91 (1994)

Proc. Nati. Acad. Sci. USA 91 (1994) 7775

FIG. 4. Comparisons of AHD2 (Left) and TH (Right) in double-labeled specimens. (a and b) Coronal section through the substantia nigra(A9) and adjoining A8. (c and d) Coronal section through the ventral tegmental area, showing A10 Qeft cell cluster, above the arrow in c, whichindicates the brain midline), and A8 (right cluster). Note the prevalence ofAHD2-containing neurons among the ventral dopaminergic population.(e andf) Section through adrenal gland; note the absence of AHD2 labeling from the bright TH-positive chromaffin cells. (Bar = 300 jm.)

and olfactory bulb. These assays demonstrate retinoic acidsynthesis in the adult striatum, mediated by AHD2, the onlyretinoic acid-generating enzyme detectable there. The exactlevels of endogenous retinoic acid remain to be determinedwith an alternate method, as the reporter cells respond poorlyto the 9-cis-retinoic acid isomer (22), which is likely to forma fraction of the retinoid content in the striatum, because ofthe high concentration of a 9-cis-retinoic acid-binding recep-tor (RXRy) here (18).

DISCUSSIONThe importance of retinoic acid as a transcriptional regulatorin the corpus striatum is evident from the extensive array ofretinoic acid receptors and binding proteins expressed in thisregion (18, 19). More than 100 genes are known to beregulated by retinoic acid (20), including that encoding theneurotrophin receptor TrkB (21). Retinoic acid levels aregenerally much higher in the embryonic than adult nervoussystem, and high levels seem to correlate with times andregions of reduced morphogenetic cell death, neuronal dif-ferentiation, and process outgrowth, but not with electricactivity (6, 22). This interpretation is consistent with rela-tively high retinoic acid levels in the olfactory bulb, which isunique in the adult brain for having to accommodate newly

formed synaptic inputs, and low levels in the hippocampus.Maybe, in the adult striatum retinoic acid is involved in somecontinuing structural adjustments necessary for acquisitionof motor patterns.The observation that the retinoic acid-generating enzyme

in the basal ganglia is present in axons and synaptic terminalsprojecting over a long distance from the midbrain reveals anunusual form of information transmission. While the princi-ple function of neuronal projections, at least in the adult, isthought to be the transfer of electrical information, axonaltransport of the retinoic acid-producing enzyme indicates away by which the mesencephalic neurons can directly exertan influence on gene transcription in the forebrain. Thisrepresents a mechanism for a long-distance trophic or induc-tive neuronal interaction.Although we describe a retinoic acid-synthesizing alde-

hyde dehydrogenase in the meso-telencephalic dopaminesystem, nonspecific aldehyde dehydrogenase activity hasbeen demonstrated in dopaminergic regions throughout thebrain (23). This is attributed to the aldehyde dehydrogenaseinvolved in dopamine metabolism. Dopamine degradation iscatalyzed mainly by a mitochondrial isoform but not thecytosolic class-1 enzyme (24), and the mitochondrial enzymeis presumably present in all dopaminergic regions, whereas

Neurobiology: McCaffery and DrAger

7776 Neurobiology: McCaffery and DrAger

FIG. 5. Coronal sections through heads of embryonic day E13 (a), E14 (b), and E16 (c) mice labeled for AHD2; relative planes of sections(a > c) move from rostral to caudal. Arrows point to the medial forebrain bundle, which at E13 (a) contains only a few AHD2-positive axons(single fibers are visible at higher magnification; not shown), and is far overshadowed by the heavy retinal labeling. At E14 (b), AHD2-positiveaxon in the medial forebrain bundle are already pronounced as compared with the AHD2-labeled optic axons in the chiasm and around thedi/mesencephalon. As in the nigrostriatal system, AHD2 is expressed in axons and growth cones ofretinal ganglion cells, but here the expressionis transient, limited to outgrowing axons and disappearing later (17). (c) Cell bodies of origin in the E16 nigrotegmental region. Not shown hereis TH labeling, which is present only in the nigrotegmnental cells and their projections but not in the retina or optic axons. (Bar = 500 inm.)AHD2 is highly expressed only in a subpopulation of dopa-minergic neurons. Nevertheless, in addition to synthesizingretinoic acid, AHD2 could function as a back-up for dopa-mine metabolism in these cells.One of the outstanding and conserved characteristics of

class 1 aldehyde dehydrogenases is their oxidation sensitivity,a property that in several other enzymes is known to form thebasis for a redox regulatory mechanism of enzymatic activity(25). It is intriguing that this oxidation-sensitive enzyme isselectively found in a neuronal system commonly linked tooxidative stress (reviewed in ref. 26). A possible pharmaco-logical correlate of the oxidation sensitivity is the susceptibil-ity to the sulfhydryl reagent disulfiram: in in vitro assays,disulfiram acts as a specific inhibitor of class 1 aldehydedehydrogenases (13). Although in vivo the drug is likely tohave additional actions due to formation of metabolites, itscentral neurotoxic effects seem to target the TH/AHD2-containing system: disulfiram, given as an alcohol deterrent(Antabuse), can lead to extrapyramidal disturbances-parkinsonism, catatonia-and basal-ganglia lesions (27-29).The loss of dopaminergic innervation to the basal ganglia

in Parkinson disease is not diffuse but follows a topographicpattern that is mimicked by the dorsoventrally graded de-generation pattern in the weaver mutant mouse, a model forparkinsonism (30). The weaver pattern resembles the distri-bution of the AHD2 projection, except for the shell of thenucleus accumbens, which is not affected in weaver. Astransplants of adrenal chromaffin cells, which in the mousedo not contain AHD2, are less successful than fetal tegmentaltransplants in Parkinson disease, presence of the enzymecould be a determining factor. Transfection of chromaffincells with a construct expressing high levels of human class1 aldehyde dehydrogenase (31) may improve the long-termtransplant success.

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