Proc. Nati. Acad. Sci. USA Vol. 91, pp. 7772-7776, August 1994 Neurobiology High levels of a retinoic acid-generating dehydrogenase in the meso-telencephalic dopamine system (bas gania/corpus siatum/nucleus accumbens/disfram/Parkluson die) PETER MCCAFFERY AND URSULA C. DRAGER Division 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 (receivedfor review January 15, 1994) ABSTRACT Retinoic acid is synthesized from retinalde- fixed, and treated with 5-bromo-4-chloro-3-indolyl f3-D- hyde by several different dehydrogenases, which are arranged galactopyranoside (X-Gal), and the calorimetric reaction is in conserved spatial and developmentally regulated patterns. quantified in an ELISA reader. The cells are much more Here we show for the mouse that a class-i aldehyde dehydro- responsive to all-trans-retinoic acid than the cis isomers, and genase, characterized by oxidation and disulfiram sensitivity, a doubling in colorimetric readings indicates a 10- to 100-fold is found in the brain at high levels only In the basal forebrain. increase in retinoic acid. We did not attempt to measure It is present in axons and terminals of a subpopulation of absolute retinoic acid levels, but all measurements shown dopaminergic neurons of the mesostriatal and meimbic represent comparisons of different samples processed in system, forming a retinoic acid-generating projection from the parallel; no values are given for the calorimetric readings, as ventral tegmentum to the corpus striatiu and the shell of the all information is contained graphically in the comparisons. nucleus accumbens. In the striatum the projection is heaviest We did the following three assays based on the reporter cells. to dorsal and rostral regions, dining gradually toward Zymography bioassay. This assay (Fig. 1) involves sepa- ventral. The enzyme is expressed early in development, shortly ration of tissue homogenates in parallel lanes by isoelectric after appearance of tyrosine hydroxylase. Other d nergic focusing, cutting the lanes into thin slices, eluting the proteins neurons in the brain, as well as the chromaffin cells of the into L15 tissue culture medium in 96-well plates, and testing for retinoic acid synthesis from 50 nM retinaldehyde in the adrenal medulla, do not contain this dehydrogenase. The presence of 2 mM NAD (5). This assay probably detects all presence ofthis enzyme may be a factor in the long-term success enzymes capable of generating any form of retinoic acid. of transplants of dopaminergic cells to the corpus stratum In Enzyme lability assay. Similar-size tissue pieces were Parkinson disease, and it may play a role in parkinsonism and dissected from the striatum and olfactory bulbs of young catatonia due to disulfiram (Antabuse) neurotoxicity. adult mice and were subjected to one of two pretreatments before being assayed for retinoic acid synthesis from added Aldehyde dehydrogenases represent a diverse family of re- retinaldehyde and NAD with the reporter cells (Fig. 2a). (i) lated enzymes involved in the oxidation of exogenous and The tissues were homogenized in L15 medium without any endogenous aldehydes (1-3). They differ in a range of char- additives or with 2.5 1LM or 12.5 ,uM disulfiram and kept on acteristics including substrate selectivity: of 13 isoforms ice, or (ii) the tissues were homogenized with or without 1 identified in the mouse, only class 1 (cytosolic) aldehyde mM dithiothreitol and kept at 370C for 10 or 20 min; the dehydrogenase was found to be capable of oxidizing retinal- control here is a 20-min air exposure at 370C in the presence dehyde to retinoic acid (4). Although in vitro this isoform can of dithiothreitol. Like the effect of air exposure, the dis- oxidize a broad range of substrates, retinoic acid production ulfiram effect can be prevented by dithiothreitol. is considered its main physiological role. In addition to class Estimates of endogenous retinoic acid levels. For this 1 aldehyde dehydrogenases, several recently reported dehy- assay (Fig. 2b), similar-size samples from adult olfactory drogenases are capable of generating retinoic acid (5). Ex- bulb, striatum, and hippocampus were homogenized in L15 pression of the different retinoic acid-generating dehydroge- medium in the presence of 2 mM NAD, but no retinaldehyde nases occurs in stereotypic spatial and developmentally was added. The homogenates were incubated overnight at regulated patterns that are similar in mammals, birds, and 370C, and the supernatants were plated in serial dilutions onto cold-blooded vertebrates. For instance, the embryonic retina the reporter cells. of all vertebrates tested contains in its dorsal part a class 1 Histology. All murine tissue, derived from an outbred mouse aldehyde dehydrogenase, termed AHD2 in the mouse, and in colony, was fixed in periodate-lysine-paraformaldehyde (9), its ventral part different dehydrogenases (5, 6). either by immersion (embryos) or by transcardiac perfusion MATERIALS AND (adult); the monkey tissue came from an adult rhesus macaque METHODS perfused with 4% paraformaldehyde for an unrelated experi- Retinoic Acid Assays. All determinations of retinoic acid ment. Cryostat sections were labeled with 2 different antisera shown here make use of a reporter cell line: teratocarcinoma to rodent class 1 aldehyde dehydrogenase (10, 11) and with a cells transfected with a sensitive retinoic acid-response ele- monoclonal antibody or an antiserum to tyrosine hydroxylase ment driving (3-galactosidase expression (7). These cells (TH; Boehringer Mannheim; Eugene Tech). Antibody binding specifically detect retinoic acid, the product of the irrevers- was visualized with fluorescent secondary antisera. ible dehydrogenation reaction of retinaldehyde, but they do not give information on the preceding enzymatic reaction, the RESULTS reversible oxidation of retinol to retinaldehyde, which is In the embryonic mouse eye, retinoic acid is generated by catalyzed by an alcohol dehydrogenase (8). The cells are four zymographically distinct enzymatic activities exposed to a retinoic acid-containing sample overnight, (Fig. la, Abbreviations: AHD2, murine class 1 (cytosolic) aldehyde dehydro- The publication costs ofthis article were defrayed in part by page charge genase; TH, tyrosine hydroxylase. payment. This article must therefore be hereby marked "advertisement" *Communication of this paper was initiated by W. J. H. Nauta and, in accordance with 18 U.S.C. §1734 solely to indicate this fact. after his death (March 24, 1994), completed by Ann M. Graybiel. 7772 Downloaded by guest on September 26, 2021 Neurobiology: McCaffery and DrAger Proc. Natl. Acad. Sci. USA 91 (1994) 7773 a AHD2 Vl V2 V3 b lower. Acidic dehydrogenases were detectable throughout the brain, but their compositions and levels varied: in most regions in both developing and mature brain, high activity was associated with the pia. In the adult brain the relatively highest activity was present in the olfactory bulbs, where several different enzymes were expressed, and only low u A levels were found in the hippocampus, which contained mainly a single activity (Fig. la, bottom traces). AHD2 was to N detectable in the brain at high levels only in one area: the r. striatum (Fig. la). Whereas in the adult striatum all retinoic acid synthesis was mediated by AHD2, the fetal striatum contained rather high levels ofacidic dehydrogenases similar to those in the embryonic eye; the basic AHD2 took over basioctory bulb slowly during the perinatal period (Fig. lb). In in vitro hippocampus assays with substrates other than retinoic acid, basic < > acidic the class 1 isoform is known to stand out from other mem- basic < > acidic bers of the family of nonspecific aldehyde dehydrogenases through its very high susceptibility to oxidation and to FIG. 1. (a) Zymographs of retinoic acid-generating enzymes in inhibition by the sulfhydryl reagent disulfiram (12, 13). The similar-size samples dissected from the corpus striatum, olfactory same bulb, and hippocampus of adult mice, processed in parallel with an distinction applies to a comparison of AHD2 with the embryonic day 16 (E16) eye, which serves here as a pH/enzyme novel retinoic acid-generating dehydrogenases, which prob- marker; the designations of the eye enzymes are indicated at the top ably do not belong to the nonspecific aldehyde dehydroge- (6). No numerical values are given for the colorimetric readings, as nase family (14): enzyme activity in homogenates of adult only the comparative values, depicted graphically, are of signifi- striatum was much more susceptible to oxidation and dis- cance. For easier comparison of enzyme activities, the colorimetric ulfiram treatment than activity in olfactory-bulb homoge- reading traces are aligned and offset. The basic enzyme activity is nates (Fig. 2a). Like the effect of air exposure, the disulfiram AHD2; the acidic activities in the brain samples represent novel effect could be prevented and, at least partially, reversed by dehydrogenases resembling those in the embryonic eye. (b) Devel- the reducing reagent dithiothreitol; it is possible that both opmental changes in retinoic acid-generating enzymes in the devel- manipulations oping striatum. Similar-size samples were dissected from the stria- target the same sulfhydryl groups on AHD2. tum of E16, postnatal day 2 (P2), and P27 mice and were processed The exact location of the enzymes in the fetal