Differential Expression of Diacylglycerol Kinase Iota and L18A

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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. The present strategy can not mechanistically link our results to function, but yields an interesting corre- lational pattern. For L18A, the rank order of expression appears to follow that of both our behavioral traits of interest, ie alcohol preference and fear-induced behavioral suppression. AA rats acquire alcohol self- administration behavior faster than Wistar rats, while ANA rats avoid alcohol.5 Furthermore, under conditions where fear of a punisher effectively suppresses behavior in both Wistar and ANA subjects, the responding of AA subjects is disinhibited. ANA subjects, on the other hand, are behaviorally inhibited even under experimental conditions which fail to sup- press behavior in the other two lines.16 In humans, a subtype of alcohol dependence has been proposed on the basis of adoption studies, thought to be charac- terized by high genetic loading, early onset, active alcohol-seeking behavior, and impaired impulse control.2,26 The behavioral characteristics of AA subjects thus parallel these traits in several important respects. A possible contribution of the L18A gene locus to this disease might therefore be worthwhile exploring. On the other hand, the expression of the DGK-like 4.3-kb transcript seems to be decreased only in the ANA rats. This locus is therefore less likely to be Figure 2 Ribosomal protein L18A mRNA expression in rat directly related to alcohol preference, and its differen- brain: (a) Autoradiogram of multiple tissue northern blot from tial expression may in fact be unrelated to behavioral total RNA hybridized with radiolabeled probe from sub- function. However, behavioral suppression under nor- cloned band No. 1/1. A single band at about 600 nt length mal, non-stressful conditions, is a striking behavioral was detected throughout all regions. Blots were re-hybridized characteristic of the ANA line. The relation of our with a ␤-actin specific probe. The diagram shows the rela- second DD-product to behavioral suppression might tively even expression level of L18A mRNA throughout dif- therefore also be worthwhile to examine. ferent regions of the brain. Lanes 1–12: (1) frontal cortex; (2) On a cellular level, both the differences in expression posterior cortex; (3) hippocampus; (4) entorhinal cortex; (5) profiles captured in the present study might be olfactory bulb; (6) striatum; (7) thalamus; (8) cerebellum; (9) midbrain; (10) pons; (11) medulla; (12) spinal cord. (b) Histo- involved in basic signal transduction mechanisms, gram of northern blot analysis demonstrating rat ribosomal such as the control of AP1 regulated genes or the regu- protein L18A mRNA levels in parietal cortex of AA and ANA lation of intracellular calcium levels. Recently, it was rats (n = 5–6/group). Values are given as percentage of found that adenylate cyclase activity is increased in the expression in naive Wistar rats (mean ± SE) and are nor- cortex of ANA rats.27 It has been postulated that protein malized for ␤-actin expression. **P Ͻ 0.001, Kruskal–Wallis kinase C and cAMP-dependent protein kinases may act ANOVA. Broken lines: 100% ± SE of expression in Wistar in parallel in synaptic facilitation, a mechanism rats. involved in sensitization.28,29 The identification of second messenger pathways involved in the development of dependence may delineate new targets for hybridization demonstrated a predominantly neuronal therapeutic approaches of these devastating diseases. expression of the DGKI-like mRNAs in discrete regions of rat brain. Strongest expression was found in the dentate gyrus and CA region of the hippocampus as well as in the piriform cortex. The gene was moderately Methods expressed in cortex, caudate putamen, thalamus and cerebellar cortex. The expression pattern seems to be Animals different from that of type IV DGK zeta, which is not Drug-naive male rats (weight ෂ300 g, standard housing expressed in caudate putamen and thalamus but seems conditions) of the following lines were used: ethanol to have higher mRNA levels in the cerebellar cortex preferring AA (Alko-Alcohol; National Public Health and is expressed also in the periphery.25 The probe Institute, Helsinki, Finland), ethanol non-preferring used for our in situ hybridization can not differentiate ANA (Alko-Non-Alcohol) and Wistar (BK Universal, between the mature and precursor transcripts, and thus Sollentuna, Sweden). All experiments were approved the intensity of the in situ signal represents a sum of by Stockholm South Animal Ethics Committee (permit these transcripts. No differences in DGKI in situ S85–89/98).

Molecular Psychiatry Expression proﬁling in brain of ethanol preferring rats W Sommer et al 106

Figure 3 Sequence and northern blot analysis of PCR product No. 5/2. (a) The sequence has a 85% similarity to human diacylglycerol kinase iota (EMBL acc. code: DGKI) sequence between position 3379 and 3817. Primer sequences are set in capital letters, the coding sequence is underlined. The open reading frame at the 5Ј-end translates to 41 amino acids, which are nearly identical to the C-terminal of the human diacylglycerol kinase iota protein. *Illustrates the valine to isoleucine substitution in the rat polypeptide. (b) Autoradiogram (partial) of multiple tissue northern blot from poly A+ RNA hybridized with radiolabeled probe from subcloned band No. 5/2 (left panel). A single band at about 4.2 kb length was detected exclusively in the lane containing the total brain sample (lanes for samples from heart, spleen, lung, liver, skeletal muscle, kidney and testis tissue not shown). Total RNA from cortex of individual rats (n = 4/group) was used and simultaneously hybridized with radiolabeled probes from subclonedbandNo.5/2andrat␤-actin gene (right panel). The autoradiogram shows two predominant mRNA species of 13 kb and 4.3 kb length and the ␤-actin control. (c) Band intensity values for the 13-kb RNA (left) and 4.3-kb RNA (right) were normalized for ␤-actin expression and given as a percentage of expression in naive Wistar rats (mean ± SE) (**P Ͻ 0.01, AA vs ANA, ANOVA). Broken lines: 100% ± SE of expression in Wistar rats (d) Ratio of 13-kb RNA/4.3-kb RNA demonstrating the reduced activity for the generation of mature 4.2-kb mRNA in ANA rats (**P Ͻ 0.01, ANA vs Wistar; °P Ͻ 0.05, ANA vs AA; ANOVA).

Molecular Psychiatry Expression profiling in brain of ethanol preferring rats W Sommer et al 107 a b 500 pmol dNTP, 10 pmol of each primer, 0.1 ␮l alpha[33P]dATP and 0.2 ␮lof50× Klen Taq polymerase mix. A master mix was prepared and 9 ␮l were added to 1 ␮l resp. 0.2-␮l aliquots of the cDNA reac- tions. The PCR was conducted in a PTC-200 DNA engine (MJ Research, Waterfront, MA, USA) under the following conditions: (1) 94°C for 5 min; (2) 40°C for 5 min; (3) 68°C for 5 min; (4) 94°C for 2 min; (5) 40°C c d for 5 min; (6) 68°C for 5 min; (7) go to step 4, 1 time; (8) 94°C for 1 min; (9) 60°C for 1 min; (10) 68°C for 2 min + 4 s per cycle; (11) go to step 8, 13 times; (12) 68°C for 7 min; (13) 4°C continuing. Sample to sample variability of the 371-bp band intensity was consist- ently below 30%. For the differential display analysis 12 anchor primers, T7-TTTTTTTTTTTTMN (AP1– AP12), and four arbitrary primers, M13r-CGAC ef TCCAAG, M13r-GCTAGCATGG, M13r-GACCATTGCA, M13r-GCTAGCAGAC (ARP1–ARP4, respectively; Hieroglyph, Genomyx, Foster City, CA, USA) were used, and PCR was run with a total of 29 cycles. Samples were loaded on a 6% acrylamide/8 M urea gel (HR-1000, Genomyx) and fractionated on a Geno- myxLR DNA sequencer. After the electrophoresis, excess urea was rinsed off, the gel transferred to What- Figure 4 In situ hybridization obtained with the antisense man 3 MM paper and dried. Autoradiography was (a, b, c, d) and the control sense (e) riboprobe generated from done overnight at room temperature. For evaluation the the subclone fragment No. 5/2, demonstrating the distribution film was realigned with the dried gel and bands that of the diacylglycerol kinase iota RNA expression in the rat brain, approximately 1.2 mm (a), −0.4 mm (b), −2.8 mm (c and appeared differentially expressed were cut out. A band e), and −9.8 mm (d) from Bregma. Panel (f) shows a high mag- was considered as differentially expressed when the nification image of the cellular distribution of the 13-kb/4.3- difference in the expression pattern occurred consist- kb mRNAs in the hippocampal CA region. CA, cornus ham- ently throughout the group. Bands appearing solely dif- monis hippocampus; Cau, caudate/putamen; Cb, cerebellum; ferent in the Wistar group were not considered in the Dg, dentate gyrus; Par, parietal cortex; Pir, piriform cortex; post differential display analysis. SHi, septohippocampal nucleus. Reamplification of differentially expressed bands Tissue preparation and RNA extraction Gel pieces were rehydrated for 10 min and then heated Following decapitation, cortical tissue was dissected to 95°C for 5 min. PCR reamplification was done for 20 out and total RNA extracted using the method of cycles with the same primer set as in the original PCR Chomczynski and Sacchi,30 washed with 75% ethanol, reaction. The PCR products were gel purified, ligated quantified spectrophotometrically, and stored at −70°C. into pGem-T (Promega) and transformed into XL1-blue For in situ hybridization brains were snap-frozen in electrocompetent cells. Plasmid DNA was prepared isopentane at −40°Candsectioned(14␮m) on a cryostat. from five to ten bacterial colonies and used for sequence analysis. For northern hybridization the Differential display RT-PCR cDNA insert was released by digestion with NotI, gel Ten-microgram aliquots of total RNA were treated with purified and labeled with alpha[32P]dCTP. For in situ 1 ␮l of RQ1 RNase-free DNase and 1 ␮l RNasin hybridization clone No. 5214 was linearized with SalI, (Promega, Madison, WI, USA) for 30 min at 37°C and and [35S]UTP labeled probes were generated by in vitro again extracted with phenol/chloroform, washed twice transcription with T7 RNA polymerase. with 70% ethanol and quantified. For cDNA synthesis 2 ␮g total RNA were incubated with 1 pmol oligo Northern blot hybridization dT(12–18) at 70°C for 3 min, and first strand synthesis Total RNA (5 ␮g) from cortical tissue of individual rats carried out with MLV reverse transcriptase (Gibco BRL, (n = 4–6/group) was loaded on a 1% agarose/2% for- Rockville, MD, USA), in a total volume of 10 ␮l for 1 h maldehyde gel and after electrophoresis in 1 × MOPS, ° at 42 C. The cDNA was diluted in H2O to a final vol- transferred to nylon membrane and immobilized by UV ume of 100 ␮l and stored at −20°C. Before the sample cross-linking. Also, pre-made multiple rat tissue north- set was used for differential display, RNA concen- ern blots containing 20 ␮g total RNA per lane (OriGene, trations of individual samples were normalized for Rockville, MD, USA) or 2 ␮g polyA+ RNA (Clontech) amplification of a cyclophilin-specific band (EMBL were used. Blots were hybridized with ෂ1.5 × 106 cpm acc-nr: RATCYCA, nucleotides 44–414). PCR was car- radiolabeled probe. The rat ␤-actin probe was obtained ried out in a total volume of 10 ␮l containing 1 ␮l10× from M Bader (Max-Delbru¨ck-Centrum, Berlin, Klen Taq buffer (Clontech, Palo Alto, CA, USA), Germany).31 After washing under high stringency con-

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