Molecular Psychiatry (2001) 6, 103–108 2001 Macmillan Publishers Ltd All rights reserved 1359-4184/01 $15.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Differential expression of diacylglycerol kinase iota and L18A mRNAs in the brains of alcohol-preferring AA and alcohol-avoiding ANA rats W Sommer1, C Arlinde1, L Caberlotto1, A Thorsell1, P Hyytia2 and M Heilig1 1NEUROTEC, Karolinska Institutet, 141 86 Huddinge, Sweden; 2Dept of Alcohol Research, National Public Health Institute, Helsinki, Finland Keywords: gene expression; Wistar rats; anxiety; alcohol- metabolism in the liver and certain brain areas com- ism; ribosomal protein; diacylglycerol kinase iota pared to the AA line.5 Ethanol preference and behavioral disinhibition in AA These studies have focused on specific pathways, eg (alcohol accepting) animals is a behavioral constellation brain reward or ethanol metabolism. Strategies of this similar to that seen in human type II alcoholism, for type require a priori knowledge or assumptions about which considerable genetic loading has been shown. In factors underlying the behaviors under study. search of novel neural substrates for this phenotype, we Recently, accumulating sequence information and compared gene expression in the cerebral cortex of the development of molecular techniques has allowed a set AA rat with two groups of control animals, the ANA of complementary strategies to be applied. These (alcohol non-accepting) line and heterogeneous Wistar attempt to identify novel candidate genes by compar- animals, by differential display RT-PCR. We identified ing expression profiles between disease phenotypes, or two transcripts, ribosomal protein L18a mRNA and 15 diacyglycerol kinase iota mRNA, which are differentially models thereof. expressed between AA and ANA rats. Ribosomal protein Here we explored the feasibility of such a tech- L18A mRNA is evenly expressed throughout the brain, nology, differential display (DD) RT-PCR, to screen for but strongly reduced in cortex of AA rats vs controls. variations in expression potentially related to alcohol Diacylglycerol kinase iota is exclusively found in the preference. We compared baseline mRNA expression brain, and expressed in a distinct regional pattern. Its profiles in the cerebral cortex of AA rats with non-alco- cortical expression is about 25% higher in AA than ANA hol preferring lines (ANA and Wistar). The three lines rats. Differential display RT-PCR seems to provide a differ in alcohol preference as well as other behavioral feasible strategy to identify previously unknown genes parameters possibly related to drug-seeking behavior, whose differential expression correlates with behavioral with a potentially useful rank order between the lines. phenotypes related to dependence. Molecular Psychiatry Ͼ (2001) 6, 103–108. For alcohol preference, this rank order is AA Wistar Ͼ ANA, while the reverse is seen for fear-induced behavioral suppression/impulse control.16 Thus, for Genetic factors play an important role in human these two behaviors of interest, the AA and ANA lines alcoholism, and alcohol preference in experimental represent potentially useful bidirectional deviations animals.1–4 Several alcohol-preferring rat lines have from ‘normality’, and may help select functionally rel- been established through selective breeding for high evant DD candidates. ethanol consumption, and used to search for potential Total RNA from parietal cortex of AA, ANA, and biological substrates of alcoholism.5–7 Early lesion Wistar rats was subjected to RT-PCR using combi- studies have shown that the cerebral cortex plays a nations of four arbitrary and 12 anchor primers. This major role in regulating alcohol self-administration in search may have covered approximately 10–15% of the rats.8 Following this finding, biochemical studies expressed genes.17 About 40 candidate bands (length identified elevated levels of noradrenaline, but sup- between 130 and 1600 bp) appeared to be differentially pressed levels of serotonin as well as dopamine in the expressed on the gels. For two bands, No. 1/1 and No. cortex of alcohol-preferring P-rats.9 More recent studies 5/2, differential expression could be confirmed (Figure have also indicated that measures of both glutamatergic 1). This outcome parallels a DD analysis of the nucleus and GABA-ergic transmission in the cerebral cortex accumbens from brains of human alcoholics and have co-segregated with ethanol preference, and are matched controls, which also covered about 10% of the also differentially affected by ethanol exposure, in two brain transcriptome, and yielded two differentially other genetic models.10,11 In the brains of AA rats, expressed genes: an increase in the 12S and 16S increased levels of dopamine and serotonin have been rRNAs, major components of functional mitochon- found.5 Differences in the opioid system between AA drial ribosomes.18 rats and their genetic counterparts, ANA rats have been In our study, band No. 1/1 contained a 130-bp reported.12–14 Also, ANA rats have a decreased ethanol sequence identical to the 3Ј-end of rat 60S ribosomal Expression profiling in brain of ethanol preferring rats W Sommer et al 104 Figure 1 (a) Autoradiogram from representative differential display gel (primer combination: AP11/ARPI) demonstrating the design of the experiment. For each strain (AA, ANA and Wistar) samples from three individual rat brains (parietal cortex) were used at two different concentrations for PCR as described in the Methods section. Panels (b) and (c) show the appearance of the two candidate bands which were confirmed for differential expression in further experiments (see Figures 2 and 3). (b) Band No. 1/1 (primers: AP1/ARP1) is not found in the AA rats. (c) Band No. 5/2 (primers: AP4/ARPI) appears to be weaker in the ANA rats. (d) RT-PCR amplification of a cyclophilin-specific band (370 bp) was used to normalize the sample set. subunit protein L18A mRNA (EMBL acc-nr: probe from subcloned PCR product No. 5/2 demon- RRRPL18A). Northern hybridization with a radiolab- strated a single transcript at 4.2 kb which is exclusively eled probe from subcloned PCR product No. 1/1 expressed in the brain tissue (Figure 3b, left). Similarly, showed a single band of approximately 600 nucleo- human DKG iota is exclusively expressed in the brain tides length, with similar intensity in several regions while DKG zeta is present also in peripheral tissues.25 of the rat brain (Figure 2a). Northern blots of total RNA Thus, it is concluded that our analysis has identified from cortical tissue of AA, ANA and Wistar rats dem- the 3Ј-end of the rat homologue of human DKG iota onstrated that L18A mRNA levels in the cortex of AA gene. rats are about seven-fold lower than those of ANA rats, Using total cortical RNA and the same probe, two with the Wistar line falling in the middle (Figure 2b). bands were identified in addition to the 4.2-kb mRNA: L18A is one of the 43 proteins forming the large sub- a major band of 13 kb size and a weaker band at about unit of the cytoplasmic ribosome.19 It is overexpressed 19 kb (Figure 3b, left). These larger RNA species most in transformed and metastatic tumor cell lines,20 but likely represent precursors of the 4.2-kb mature mRNA- its function within the ribosome is unknown. Changes transcript. Using ␤-actin as an internal standard, only in its expression might contribute to altered protein the 4.2 mRNA species was significantly reduced in the synthesis. Furthermore, several ribosomal proteins ANA rats compared to AA animals (Figure 4c). Similar have been found to operate outside the ribosome com- results were obtained with a GADPH specific probe as plex.21 L18A contains two zipper-like domains and internal control (data not shown). Furthermore, the probably binds to the inducible transcription factor c- ratio of 4.2 kb/13 kb RNA in ANA rats is significantly jun.22 Therefore, the striking difference in the different from both other lines (Figure 4d). This calcu- expression of L18A mRNA in the AA and ANA lines lation confirms a true difference in 4.2-kb mRNA lev- may also have importance for cellular regulation of c- els, since it eliminates the possible influence of the jun/AP1 controlled genes. internal control, which by itself could be differentially The 640-bp sequence of band No. 5/2 has a strong expressed between the lines. As a possible explanation similarity (85%) to the 3Ј-end of the coding region of for the differences in 4.2-kb/13-kb RNA ratio we pro- human diacylglycerol kinase iota (EMBL acc-nr: DGKI; pose that these represent different splicing activities in Figure 3a). In fact, the 5Ј-end of No. 5/2 contains a the three rat strains, which may provide a mechanism reading frame of 41 amino acids. The resulting poly- to regulate DGK iota activity in the brain. peptide is nearly identical to the C-terminus of human Diacylglycerol is the key activator of protein kinase diacylglycerol kinase iota (Val to Iso substitution in C. The various DGKs phosphorylate diacylglycerol to position 31). It contains one of the ankyrin-like repeats phosphatic acid, and therefore act as negative regu- characteristic for DGK isotypes iota and zeta, which lators of protein kinase C mediated second messenger together comprise type IV of the DGK family.23 The pathways.23 At least nine mammalian isotypes of ankyrin-like repeats are important for the nuclear local- diacylglycerol kinase exist and are expressed in the ization of the enzyme.24 brain at varying levels. The molecular diversity and Northern blot analysis using polyA+ RNA from total distinct expression pattern of DGK isotypes suggests an brain and seven peripheral tissues with radiolabeled importance of these enzymes for brain function. In situ Molecular Psychiatry Expression profiling in brain of ethanol preferring rats W Sommer et al 105 hybridization signals could thus be observed between AA, ANA and Wistar rats.