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In situ hybridization of dihydroxyacetone phosphate acyltransferase, the regulating enzyme involved in plasmalogen biosynthesis
Molecular Brain Research 136 (2005) 142 – 147 www.elsevier.com/locate/molbrainres
Research report
In situ hybridization of dihydroxyacetone phosphate acyltransferase, the regulating enzyme involved in plasmalogen biosynthesis Agne`s Andre´a,1, Christian Tessiera,b, Lionel Bre´tillona, Jean-Louis Se´be´dioa, Jean-Michel Chardignya,T a
INRA, Unite´ de Nutrition Lipidique, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France b UPRES Lipides et Nutrition, Universite´ de Bourgogne, Dijon, France Accepted 14 January 2005 Available online 8 March 2005
Abstract In situ hybridization can be carried out using different methods. The experimenter has to choose various parameters: the type of tissue fixation, the time of incubation, and the duration of the exposure time. All these parameters are determinant for the sensitivity and the resolution of this technique. This publication of technical aspects described different experiments performed for in situ hybridization on liver tissue. We may conclude on the parameters to optimize each step of the hybridization procedure. Moreover, this technique could be transposed to the brain and applied to little structures with a light expression of DHAP-AT. D 2005 Elsevier B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing, and imaging techniques Keywords: In situ hybridization; Dihydroxyacetone phosphate acyltransferase; Plasmalogen; Liver; Brain
Among the glycerophospholipids (Gpl), plasmalogens (PlsGpl) represent a special subclass distinguished by their vinyl–ether bond at the sn-1 position of the glycerol backbone. The first two steps of PlsGpl biosynthesis are carried out in peroxisomes. Dihydroxyacetone phosphate acyltransferase (DHAP-AT), an enzyme localized at the internal surface of the peroxisomal membrane, catalyzes the first step which consists on the esterification of the free hydroxyl group of DHAP by a long chain acyl coenzyme A to form acyl-DHAP. PlsGpl are widely distributed in all mammalian tissues and body fluids. Their high content in heart and brain suggests that they play an important role in the structure and function of biological membranes. An interesting aspect of PlsGpl is their putative role in signal T Corresponding author. Fax: +33 3 80 69 32 23. E-mail address: [email protected] (J.-M. Chardigny). 1 Funded by a fellowship from INRA and the Regional Council of Burgundy. 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.01.018
transduction. They are known to be reservoirs of polyunsaturated fatty acids (PUFA). Indeed, they predominantly contain at their sn-2 position arachidonic or docosahexaenoic acids which are released after stimulation [4]. Another potential role of PlsGpl has been suggested by Engelmann et al. [3]: these compounds may protect cells against oxidative stress. Indeed, their vinyl–ether bond is preferentially oxidized and as a consequence protecting PUFA at the sn-2 position from oxidation. Moreover, the important role of PlsGpl in humans is emphasized by their deficiency in peroxisomal disorders. They are strongly deficient in tissues and cells of patients suffering from cerebro-hepato-renal syndrome (Zellweger syndrome, ZS), in which functional peroxisomes are absent. These findings suggest that at least one of the enzymes involved in PlsGpl biosynthesis is deficient in patients with ZS and specially one involved in the introduction of the ether bond [9]. Schutgens et al. [10] have shown that PLsGpl synthesis is affected because of a defect in DHAP-AT. Exposure of
A. Andre´ et al. / Molecular Brain Research 136 (2005) 142–147
cultured skin fibroblasts from patients with ZS to hexadecylglycerol, a plasmalogen precursor, partially restored PlsGpl contents [12]. In the liver, DHAP-AT localizes exclusively in peroxisomes [11]. Given its high content in these organelles, the in situ hybridization of DHAP-AT was developed on the liver. The brain was then studied because of its high level of plasmalogens (between 1/2 and 2/3 of the ethanolamine phospholipids are plasmalogens [8]. Moreover, contrary to the liver, which presents an homogeneous structure, the brain has a complex organization. The cerebral cortex is the outer layer of the cerebral hemispheres of the brain. It mediates all conscious activity including planning, problem solving, language, executive function, and speech. It is also involved in perception (hearing, vision) and voluntary motor activity. The cerebellum is a large cauliflower-looking structure on the top of the brainstem whose cortex is covered of gray matter. This structure is very important in motor movement for coordinating voluntary movements (walking, posture, speech) and in learning. The hippocampus is a thin structure in the subcortex shaped like a seahorse. The hippocampus is important for converting short-term memory to more permanent memory [2] and for recalling spatial relationships in the world about us. All these structures have specific, essential functions and different plasmalogen contents [7]. In this context, we developed a method for studying the localization of the DHAP-AT mRNA in the brain. After validation on the liver, the technique of in situ hybridization was then applied and optimized on different brain structures in order to illustrate the interest of this method. The final aim of this work was to study the tissue localization of the DHAP-AT mRNA in the brain.
1. Materials and methods 1.1. Animals Wistar males rats (Janvier’s bredding, Le Genest-St-Isle, France) were housed in animal quarters under controlled temperature (21 F 1 8C) and light conditions (12-h light/12h dark cycle). They had free access to food and water. All animal use and care were conducted according to the European legislation (authorization A 21200 for the animals housing and personal authorization 21 CAE 056 for experimentation on animals). 1.2. Preparation of the slides 1.2.1. Experiment 1 Animals were anesthetized with intraperitoneal injection of pentobarbital (500 AL/500 g body weight) and then rapidly perfused transcardially with 0.9% saline, followed by 10% paraformaldehyde in 0.9% NaCl buffer at 4 8C for 20 min. The liver and brain were removed, frozen in cold isopentane, and then stored at 80 8C until used.
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1.2.2. Experiment 2 After fixation with 4% paraformaldehyde in 0.9% NaCl as described in experiment 1, brains were postfixed for 3 h at 4 8C in the same solution of paraformaldehyde. They were then placed in 15% sucrose diluted in phosphate buffer (0.1 M) for 3 h at 4 8C. Finally, brain was frozen in cold isopentane and then stored at 80 8C until used. 1.2.3. Experiment 3 Rat liver and brain were rapidly removed and frozen in cold isopentane and stored at 80 8C until used. After these three different experiments, sagittal sections from the frontal cortex to the cerebellum were cut on a cryomicrotome (Reichert-Jung) with a thickness of 8 or 14 Am, mounted on SuperFrost Plus slides, and stored at 80 8C. Liver slides were obtained in the same way. 1.3. Brain RNA preparation Total RNA was prepared using TRIzolR Reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, total RNA was extracted with chloroform followed by 2-propanol precipitation. The pellet was washed in 75% ethanol, dried, and dissolved in diethylpyrocarbonate (DEPC) water. The obtained RNA was quantified spectrophotometrically by measuring the absorbance at 260 nm. 1.4. Generation of cDNA from brain RNA The rat DHAP-AT sequence used was from the literature (GenBank accession no NM_053410). PCR primers were selected with a computer program (NCBI, primer) for amplification of a 405 bp sequence. PCR primers were purchased from MWG-Biotech AG: forward primer CGT GTT TGC CCG TCC TT and reverse primer AGC GCG TCG TAA CAC TGA. RT-PCR was performed with a thermal cycler (Bio-Rad) using 1 Ag of RNA in 25 AL of the reaction mixture consisting of 2.5 U of AMV reverse transcriptase, 225 pmol of each primer, 0.5 AL of dNTP mix (10 mM), 1 AL of MgSO4 (25 mM), 5 AL of AMV 5 Reaction buffer, and 2.5 U of Tfl DNA polymerase. RNA was reverse transcribed for 45 min at 48 8C. After initial denaturation at 94 8C for 2 min, the reaction mixture was subjected to 40 cycles of 1 min denaturation at 94 8C, 1 min annealing at 53.1 8C, and 1.5 min extension at 72 8C, followed by a final 10 min extension step at 72 8C. PCR-derived fragments were separated by one agarose gel electrophoresis (0.7%) and stained with ethidium bromide. The fragment of interest was excised, extracted from the agarose gel and purified (Qiagen, gel extraction Kit). PCR products were subcloned into the pGEMR-T easy vector (3015 bp, Promega). JM 109-competent cells were then transformed with this vector. A positive clone was selected for a last amplification. Plasmids were then purified using QIAfilter Plasmid Maxi Kits (Qiagen) and sequenced using a ceq 2000 Beckman Coulter.
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A. Andre´ et al. / Molecular Brain Research 136 (2005) 142–147
1.5. Riboprobe synthesis The plasmids containing a rat cDNA fragment of 405 bp were linearized with BstX I and Apa I for the antisense and sense probes, respectively. Radioactive cRNA copies were synthesized using the Riboprobe in vitro transcription system (Promega). The mixture consisted on 4 AL of transcription optimized 5 buffer, 0.5 Ag of linearized plasmid, 2 AL of 100 mM DTT, 1.5 AL of 10 mM ATP, GTP, and CTP, 50 ACi of ~-35S-UTP, 20 U of recombinant Rnasin ribonuclease inhibitor, and 20 U of either T7 (antisense probe) or SP6 (sense probe) RNA polymerase. This mixture was incubated for 45 min at 37 8C, then 6 min after the addition of 1 U RQ1 Dnase. Radioactive RNA was extracted with 100 AL of a HENS buffer containing 20 mM HEPES (pH 7.0), 5 mM EDTA, 50 mM NaCl, 0.1% SDS, and tRNA (1 Ag/AL). This last compound removed unincorporated nucleotides. The RNA was precipitated 30 min at 80 8C by adding 65 AL of 7.5 M ammonium acetate and 500 AL of cold absolute ethanol. After centrifugation (20 min, 15,000 g), the pellet was dried, redissolved in HENS buffer, and then heated at 65 8C for 1 min. This final solution of radioactive RNA was counted. 1.6. Hybridization 1.6.1. Experiment 1 Slides were placed in paraformaldehyde for 15 min on ice, then rinsed two times in PBS for 5 min. They were dipped in 0.25% anhydric acetic acid/0.1 M TEA for 10 min and rinsed in 2 SSC (0.3 M NaCl, 30 mM sodium citrate) for 20 s. Slides were dehydrated with ethanol and successively put in 5 mM MgCl2–PBS for 10 min, in 0.25 M Tris–0.1 M glycine for 10 min, and in 50% formamide-2 SET (0.3 M NaCl, 60 mM Tris–HCl, 4 mM EDTA, pH 8.0) at 37 8C for 10 min. The hybridization mixture consisting of 7.25 mL sterile water, 2.5 mL 20 SET, 0.1 g PVP (0.1 AM), 0.1 g Ficoll (0.01 M), 0.1 g BSA, 12.5 mL formamide (13 M), 4 g dextran sulfate, 50 mg/ mL tRNA, and 500 AL DTT (1 M) was prepared with ~-35S-labeled sense or antisense probes to obtain a concentration of 2.107 cpm/mL. These solutions (sense or antisense) were spotted on slides and a coverslip was placed on the sections. Slides were incubated at 45 8C for 4 h in a humid hybridization chamber. After incubation, the slides were washed rapidly by submersion in 4 SSC, then in 50% formamide-2 SET-0.04% mercaptoethanol for 15 min at 60 8C and lastly in 4 SSC. Slides were incubated in Rnase A solution (10 mg/mL)-3 SET at 37 8C for 20 min and rinsed in 1 SSC at room temperature for 30 min with a light agitation. The last wash consisted of 0.1% mercaptoethanol in 0.2 SET at 50 8C for 30 min. Slides were dehydrated in 30%, 50%, 70% ethanol containing 0.3 M ammonium acetate, and after in 85%, 95%, and 100% ethanol. The hybridized sections were dried and then autoradiographed. Slides were dipped in Hypercoat LM-1 emulsion (Amersham) and exposed for 30 days at 4 8C. At the time of
developing, slides were put in D-19 developer (Kodak) for 2.5 min at 15 8C, rinsed, fixed (fixer Kodak) for 5 min, and placed in water for 10 min. Slides were colored with Harris’ hematoxylin and aqueous eosin. 1.6.2. Experiment 2 Slides were incubated at 45 8C for 6 h instead of 4 h. 1.6.3. Experiment 3 Slides were incubated overnight at 45 8C in a humid hybridization chamber. The succession of wash was different. After incubation, the slides were washed rapidly by submersion in 2 SSC, then incubated for 1 h in 200 mL of 2 SSC with 1 mL of 1 M DTT. Slides were then placed in 50% formamide-2 SSC-10 mM DTT for 20 min at 60 8C and immediately after rinsed in 3 baths of 2 SSC for 3 min each at room temperature. Slides were washed in 2 SSC for 1 h with a light agitation. The last wash consisted on 0.1 SSC for 30 min. The end of the experiment was the same as in experiment 1. 1.7. Exposure time Slides were processed following experiment 1 and then exposed for 20, 30, 40, or 60 days at 4 8C before developing.
2. Results 2.1. cDNA characterization As the probe for the DHAP-AT RNA was not commercially available, it was synthesized and then sequenced for control. The sequence (Fig. 1) of the synthesized cDNA was compared with that of the Rattus norvegicus acyl-CoA DHAP-AT gene (GenBank accession no NM_053410). A total homology was found with a part of the coding sequence between the 1473 and 1877 nucleotides. This synthesized 405-bp probe was used for all the experiments. 2.2. Choice of the optimal parameters for the hybridization (data not shown) Experiments 1 and 2 were tested because the fixation of tissues is often necessary to preserve the cell structure. However, experiment 3, faster, gave satisfactory results and was finally chosen. We considered that a step of fixation was not necessary in our case because we did not study a localization of mRNA at the ultrastructural level but only at the structural level. Moreover, the thinner the slide is, the better the observation will be; the best results were obtained with 8-Am-thickness slides. The composition of the hybridization buffer must provide optimal conditions for the formation of hybrids at the
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Fig. 1. Nucleotide sequence of DHAP-AT probe. A 405-bp PCR product generated from brain RNA with specific primers (underlined) to the rat DHAP-AT was sequenced. The numbers beside the two nucleotides highlighted represent their positions from the beginning of the DHAP-AT gene initially cloned.
incubation temperature. Dextran sulfate was added to accelerate the speed of hybridization process. Ficoll and polyvinylpyrrolidone, high molecular mass molecules, favor the hybridization. BSA decrease electrostatic bonds. tRNA was added in order to reduce non-specific adsorption between the probe and the tissue section. The duration of incubation was also tested (experiment 1 vs. 2 and 3). A long period of incubation was not necessary in our experiments as illustrated by the lack of improvement of the intensity of the signal. Moreover, a short period of incubation is preferable because tissues can be damaged and dried if the humidity of the chamber is not well controlled. The technique developed gave qualitative but not quantitative results; as a consequence, it was not useful to increase the duration of incubation. The time of wash and agitation are two critical parameters because they determine the intensity of the background. Indeed, washes eliminate the aspecific hybrids and also aspecific adsorption between the probe and the different components of the cell. In experiment 1, the total wash time was 95 min whereas it was 180 min in experiment 3. The background was insignificant in the first experiment that’s why we considered that this time of wash was sufficient and it was not necessary to prolong it. For the liver, an exposure of 20 days appeared to be sufficient to reveal a good signal. However, for brain slides, an exposure of 30 days was necessary. Longer exposures were useless. The exposure time must be adapted for each structure because of the level of hybridization. The following results correspond to the experiment made in the optimal conditions.
2.3. In situ hybridization on liver (Fig. 2) The radioactive signal for the sense probe, characterizing the background, was very weak (Fig. 2C). This confirmed the specificity of the antisense probe, the signal being specific of the DHAP-AT mRNA. In the liver (Fig. 2A), the signal of the antisense was very high and homogeneous. 2.4. In situ hybridization on brain (Fig. 3) The radioactive signal of the sense probe was as weak in the brain as in the liver (Figs. 3C1, C2, and C3). In the brain, the signal of the antisense was weaker and different in each structure. In the frontal cortex (Fig. 3A2), the in situ hybridization did not revealed any expression of DHAP-AT. The comparison of some images in contrast phase vs. dark field showed a specific radioactivity localization in the area of the hippocampus (Fig. 3A1) and in the cerebellum (Fig. 3A3). The signal was especially high in the internal structure of the cerebellum, in the hippocampus, and in the corpus callosum.
3. Discussion The aim of this work was to develop in situ hybridization of DHAP-AT mRNA. The liver was chosen because of the high expression and activity of the studied enzyme and allowed to validate the technique. Indeed, the background is very weak and even non-existent, whereas the specific signal of the DHAP-AT mRNA was very high. The optimal
Fig. 2. In situ hybridization of DHAP-AT in the liver. A and B correspond to an observation of the hybridization with the antisense probe in dark field or contrast phase, respectively. C and D correspond to an observation of the hybridization with the sense probe in dark field or contrast phase, respectively.
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Fig. 3. In situ hybridization of DHAP-AT in the brain. 1, 2, and 3 correspond to observations made in the area of the hippocampus, in the cortex and in the cerebellum, respectively. A and B correspond to an observation of the hybridization with the antisense probe in dark field or contrast phase, respectively. C and D correspond to an observation of the hybridization with the sense probe in dark field or contrast phase, respectively. All images were taken with a 100 objective using an Axiovert 25 Zeiss microscope.
conditions determined for in situ hybridization on the liver were a 4-h incubation period and a 95-min wash period. Slides were then exposed for 20 days before developing. The high signal in this tissue may be explained by the high content of peroxisomes, subcellular organelles known to contain the DHAP-AT [11]. Moreover, this high expression is closely related with a high enzymatic activity (data not shown). The liver produces PlsGpl, does not accumulate them (less than 5% of the ethanolamine phospholipids are the plasmalogen form), but secretes nascent lipoproteins in which 20–30% of the ethanolamine phospholipids are plasmenylethanolamine [13]. The PlsGpl synthesis and exportation are both important. Such information may explain the paradox between the high expression of DHAP-AT in the liver and its low level of PlsGpl. This method is of great interest because it can be also transposed on little structures in order to determine a specific localization of the DHAP-AT mRNA and on structures with a light level of expression.
By way of illustration, in situ hybridization was performed on brain with a particular focus on white vs. gray matter and also on some specific structures like cerebellum, hippocampus, and cortex. Indeed, the brain has a complex organization and a particular structure. It is composed of white and gray matters: about 40% of the human brain is made up of gray matter whereas 60% is white matter. The white matter contains lots of nerve fibers that are ensheathed in a membrane called myelin. About 40% of the ethanolamine glycerophospholipids are plasmalogens in human gray matter [5] and 70% in white matter. We can suppose that in a plasmalogen-rich structure, the expression of DHAP-AT gene is intense which leads to a high level of mRNA. The technique used on the liver was applied on the brain with only one little adaptation: the duration of exposure was prolonged to 30 days for optimal results. The expression of DHAP-AT in the brain appeared weak in contrast with its intense expression in the liver. In the liver, a major function of plasmalogen biosynthetic enzymes may be the provision of
A. Andre´ et al. / Molecular Brain Research 136 (2005) 142–147
plasmalogen for secretion into lipoproteins. Brain plasmalogens cannot be of liver origin because entire phospholipids cannot go through the hematoencephalic barrier. In the brain, DHAP-AT expression is weaker than in the liver but may be sufficient to synthesize and maintain a plasmalogen pool. We identified in the rat brain a structure–localization relationship. Indeed, our results showed a high signal in the internal structure of the cerebellum, in the hippocampus and in the corpus callosum. This latter structure is a thick band of nerve fibers located between the cerebral hemispheres. It is an oval-shaped center of white substance, surrounded on all sides by a narrow convoluted margin of gray matter, which presents an equal thickness in nearly every part. Fibers of the corpus callosum contain lots of myelinated axons that is why this structure is white. In the same way, the cerebellum contains more white matter than the other areas and it is known that white matter, whose myelin represents the main constituent, is especially rich in PlsGpl. These two marked structures are both rich in myelin and in plasmalogens. Such information may easily explain results obtained by in situ hybridization. Other peroxisomal proteins and their mRNA have been localized in the white matter [6,14]. Such information are in concordance with our results: DHAP-AT is a peroxisomal protein and its mRNA is mainly localized in white matter. Marked structures are rich in myelin. Oligodendrocytes are the myelin-forming cells of the central nervous system [1]. Enzymes for the synthesis of myelin glycerophospholipids are found in the oligodendrocyte cytoplasm that is why we may suppose that DHAP-AT localizes in these cells too. In the frontal cortex, the in situ hybridization did not revealed any expression of DHAP-AT. However, this structure contains plasmalogens [7]. We can hypothesize that the level of DHAP-AT expression is too weak to be detected by the method used. In conclusion, all experiments performed allowed us to determine the optimal conditions to use for in situ hybridization on the brain. This technique is a very powerful technique especially for the specific cellular and/or tissue localization of particular gene expression. Moreover, it is the only technique which allows a visualization in situ of mRNA. It appeared sensitive enough to be used on little structures with a light expression of the studied enzyme. However, a major disadvantage of this technique is the difficulty to assess quantitative differences. For a precise quantification, it is necessary to have recourse to PCR or Northern blotting. DHAP-AT mRNA are localized all over the liver whereas the expression within the brain seems to be specific to structures rich in white matter (the corpus callosum and the internal structure of the cerebellum), which contain plasmalogen-rich myelin. Studies concerning
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the effect of fatty acids on the regulation of the DHAP-AT expression in the brain are in progress.
Acknowledgments The authors gratefully acknowledge MA Maire for her technical assistance. A Tessier is acknowledged for her interest to the study and valuable comments.
References [1] N. Baumann, D. Pham-Dinh, Biology of oligodendrocyte and myelin in the mammalian central nervous system, Physiol. Rev. 81 (2001) 871 – 927. [2] H. Eichenbaum, T. Otto, N.J. Cohen, The hippocampus-what does it do? Behav. Neural Biol. 57 (1992) 2 – 36. [3] B. Engelmann, Plasmalogens: targets for oxidants and major lipophilic antioxidants, Biochem. Soc. Trans. 32 (2004) 147 – 150. [4] A.A. Farooqui, L.A. Horrocks, Plasmalogens, phospholipase A2, and docosahexaenoic acid turnover in brain tissue, J. Mol. Neurosci. 16 (2001) 263 – 284. [5] L.A. Horrocks, Content, composition, and metabolism of mammalian and avian lipids that contain ether groups, in: F. Snyder (Ed.), Ether Lipids: Chemistry and Biology, Academic Press, New York, 1972, pp. 177 – 272. [6] S. Houdou, S. Takashima, Y. Suzuki, Immunohistochemical expression of peroxisomal enzymes in developing human brain, Mol. Chem. Neuropathol. 19 (1993) 235 – 248. [7] M. Kubota, M. Nakane, T. Nakagomi, A. Tamura, H. Hisaki, H. Shimasaki, N. Ueta, Regional distribution of ethanolamine plasmalogen in the hippocampal CA1 and CA3 regions and cerebral cortex of the gerbil, Neurosci. Lett. 301 (2001) 175 – 178. [8] N. Nagan, R.A. Zoeller, Plasmalogens: biosynthesis and functions, Prog. Lipid Res. 40 (2001) 199 – 229. [9] G. Schrakamp, R.B. Schutgens, R.J. Wanders, H.S. Heymans, J.M. Tager, H. Van den Bosch, The cerebro-hepato-renal (Zellweger) syndrome. Impaired de novo biosynthesis of plasmalogens in cultured skin fibroblasts, Biochim. Biophys. Acta 833 (1985) 170 – 174. [10] R.B. Schutgens, G.J. Romeyn, R.J. Wanders, H. van den Bosch, G. Schrakamp, H.S. Heymans, Deficiency of acyl-CoA: dihydroxyacetone phosphate acyltransferase in patients with Zellweger (cerebrohepato-renal) syndrome, Biochem. Biophys. Res. Commun. 120 (1984) 179 – 184. [11] H. Singh, K. Beckman, A. Poulos, Exclusive localization in peroxisomes of dihydroxyacetone phosphate acyltransferase and alkyl-dihydroxyacetone phosphate synthase in rat liver, J. Lipid Res. 34 (1993) 467 – 477. [12] R. Styger, U.N. Wiesmann, U.E. Honegger, Plasmalogen content and beta-adrenoceptor signalling in fibroblasts from patients with Zellweger syndrome. Effects of hexadecylglycerol, Biochim. Biophys. Acta 1585 (2002) 39 – 43. [13] J.E. Vance, Lipoproteins secreted by cultured rat hepatocytes contain the antioxidant 1-alk-1-enyl-2-acylglycerophosphoethanolamine, Biochim. Biophys. Acta 1045 (1990) 128 – 134. [14] K. Zaar, H.P. Kost, A. Schad, A. Volkl, E. Baumgart, H.D. Fahimi, Cellular and subcellular distribution of d-aspartate oxidase in human and rat brain, J. Comp. Neurol. 450 (2002) 272 – 282.
Research report
In situ hybridization of dihydroxyacetone phosphate acyltransferase, the regulating enzyme involved in plasmalogen biosynthesis Agne`s Andre´a,1, Christian Tessiera,b, Lionel Bre´tillona, Jean-Louis Se´be´dioa, Jean-Michel Chardignya,T a
INRA, Unite´ de Nutrition Lipidique, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France b UPRES Lipides et Nutrition, Universite´ de Bourgogne, Dijon, France Accepted 14 January 2005 Available online 8 March 2005
Abstract In situ hybridization can be carried out using different methods. The experimenter has to choose various parameters: the type of tissue fixation, the time of incubation, and the duration of the exposure time. All these parameters are determinant for the sensitivity and the resolution of this technique. This publication of technical aspects described different experiments performed for in situ hybridization on liver tissue. We may conclude on the parameters to optimize each step of the hybridization procedure. Moreover, this technique could be transposed to the brain and applied to little structures with a light expression of DHAP-AT. D 2005 Elsevier B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing, and imaging techniques Keywords: In situ hybridization; Dihydroxyacetone phosphate acyltransferase; Plasmalogen; Liver; Brain
Among the glycerophospholipids (Gpl), plasmalogens (PlsGpl) represent a special subclass distinguished by their vinyl–ether bond at the sn-1 position of the glycerol backbone. The first two steps of PlsGpl biosynthesis are carried out in peroxisomes. Dihydroxyacetone phosphate acyltransferase (DHAP-AT), an enzyme localized at the internal surface of the peroxisomal membrane, catalyzes the first step which consists on the esterification of the free hydroxyl group of DHAP by a long chain acyl coenzyme A to form acyl-DHAP. PlsGpl are widely distributed in all mammalian tissues and body fluids. Their high content in heart and brain suggests that they play an important role in the structure and function of biological membranes. An interesting aspect of PlsGpl is their putative role in signal T Corresponding author. Fax: +33 3 80 69 32 23. E-mail address: [email protected] (J.-M. Chardigny). 1 Funded by a fellowship from INRA and the Regional Council of Burgundy. 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2005.01.018
transduction. They are known to be reservoirs of polyunsaturated fatty acids (PUFA). Indeed, they predominantly contain at their sn-2 position arachidonic or docosahexaenoic acids which are released after stimulation [4]. Another potential role of PlsGpl has been suggested by Engelmann et al. [3]: these compounds may protect cells against oxidative stress. Indeed, their vinyl–ether bond is preferentially oxidized and as a consequence protecting PUFA at the sn-2 position from oxidation. Moreover, the important role of PlsGpl in humans is emphasized by their deficiency in peroxisomal disorders. They are strongly deficient in tissues and cells of patients suffering from cerebro-hepato-renal syndrome (Zellweger syndrome, ZS), in which functional peroxisomes are absent. These findings suggest that at least one of the enzymes involved in PlsGpl biosynthesis is deficient in patients with ZS and specially one involved in the introduction of the ether bond [9]. Schutgens et al. [10] have shown that PLsGpl synthesis is affected because of a defect in DHAP-AT. Exposure of
A. Andre´ et al. / Molecular Brain Research 136 (2005) 142–147
cultured skin fibroblasts from patients with ZS to hexadecylglycerol, a plasmalogen precursor, partially restored PlsGpl contents [12]. In the liver, DHAP-AT localizes exclusively in peroxisomes [11]. Given its high content in these organelles, the in situ hybridization of DHAP-AT was developed on the liver. The brain was then studied because of its high level of plasmalogens (between 1/2 and 2/3 of the ethanolamine phospholipids are plasmalogens [8]. Moreover, contrary to the liver, which presents an homogeneous structure, the brain has a complex organization. The cerebral cortex is the outer layer of the cerebral hemispheres of the brain. It mediates all conscious activity including planning, problem solving, language, executive function, and speech. It is also involved in perception (hearing, vision) and voluntary motor activity. The cerebellum is a large cauliflower-looking structure on the top of the brainstem whose cortex is covered of gray matter. This structure is very important in motor movement for coordinating voluntary movements (walking, posture, speech) and in learning. The hippocampus is a thin structure in the subcortex shaped like a seahorse. The hippocampus is important for converting short-term memory to more permanent memory [2] and for recalling spatial relationships in the world about us. All these structures have specific, essential functions and different plasmalogen contents [7]. In this context, we developed a method for studying the localization of the DHAP-AT mRNA in the brain. After validation on the liver, the technique of in situ hybridization was then applied and optimized on different brain structures in order to illustrate the interest of this method. The final aim of this work was to study the tissue localization of the DHAP-AT mRNA in the brain.
1. Materials and methods 1.1. Animals Wistar males rats (Janvier’s bredding, Le Genest-St-Isle, France) were housed in animal quarters under controlled temperature (21 F 1 8C) and light conditions (12-h light/12h dark cycle). They had free access to food and water. All animal use and care were conducted according to the European legislation (authorization A 21200 for the animals housing and personal authorization 21 CAE 056 for experimentation on animals). 1.2. Preparation of the slides 1.2.1. Experiment 1 Animals were anesthetized with intraperitoneal injection of pentobarbital (500 AL/500 g body weight) and then rapidly perfused transcardially with 0.9% saline, followed by 10% paraformaldehyde in 0.9% NaCl buffer at 4 8C for 20 min. The liver and brain were removed, frozen in cold isopentane, and then stored at 80 8C until used.
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1.2.2. Experiment 2 After fixation with 4% paraformaldehyde in 0.9% NaCl as described in experiment 1, brains were postfixed for 3 h at 4 8C in the same solution of paraformaldehyde. They were then placed in 15% sucrose diluted in phosphate buffer (0.1 M) for 3 h at 4 8C. Finally, brain was frozen in cold isopentane and then stored at 80 8C until used. 1.2.3. Experiment 3 Rat liver and brain were rapidly removed and frozen in cold isopentane and stored at 80 8C until used. After these three different experiments, sagittal sections from the frontal cortex to the cerebellum were cut on a cryomicrotome (Reichert-Jung) with a thickness of 8 or 14 Am, mounted on SuperFrost Plus slides, and stored at 80 8C. Liver slides were obtained in the same way. 1.3. Brain RNA preparation Total RNA was prepared using TRIzolR Reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, total RNA was extracted with chloroform followed by 2-propanol precipitation. The pellet was washed in 75% ethanol, dried, and dissolved in diethylpyrocarbonate (DEPC) water. The obtained RNA was quantified spectrophotometrically by measuring the absorbance at 260 nm. 1.4. Generation of cDNA from brain RNA The rat DHAP-AT sequence used was from the literature (GenBank accession no NM_053410). PCR primers were selected with a computer program (NCBI, primer) for amplification of a 405 bp sequence. PCR primers were purchased from MWG-Biotech AG: forward primer CGT GTT TGC CCG TCC TT and reverse primer AGC GCG TCG TAA CAC TGA. RT-PCR was performed with a thermal cycler (Bio-Rad) using 1 Ag of RNA in 25 AL of the reaction mixture consisting of 2.5 U of AMV reverse transcriptase, 225 pmol of each primer, 0.5 AL of dNTP mix (10 mM), 1 AL of MgSO4 (25 mM), 5 AL of AMV 5 Reaction buffer, and 2.5 U of Tfl DNA polymerase. RNA was reverse transcribed for 45 min at 48 8C. After initial denaturation at 94 8C for 2 min, the reaction mixture was subjected to 40 cycles of 1 min denaturation at 94 8C, 1 min annealing at 53.1 8C, and 1.5 min extension at 72 8C, followed by a final 10 min extension step at 72 8C. PCR-derived fragments were separated by one agarose gel electrophoresis (0.7%) and stained with ethidium bromide. The fragment of interest was excised, extracted from the agarose gel and purified (Qiagen, gel extraction Kit). PCR products were subcloned into the pGEMR-T easy vector (3015 bp, Promega). JM 109-competent cells were then transformed with this vector. A positive clone was selected for a last amplification. Plasmids were then purified using QIAfilter Plasmid Maxi Kits (Qiagen) and sequenced using a ceq 2000 Beckman Coulter.
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1.5. Riboprobe synthesis The plasmids containing a rat cDNA fragment of 405 bp were linearized with BstX I and Apa I for the antisense and sense probes, respectively. Radioactive cRNA copies were synthesized using the Riboprobe in vitro transcription system (Promega). The mixture consisted on 4 AL of transcription optimized 5 buffer, 0.5 Ag of linearized plasmid, 2 AL of 100 mM DTT, 1.5 AL of 10 mM ATP, GTP, and CTP, 50 ACi of ~-35S-UTP, 20 U of recombinant Rnasin ribonuclease inhibitor, and 20 U of either T7 (antisense probe) or SP6 (sense probe) RNA polymerase. This mixture was incubated for 45 min at 37 8C, then 6 min after the addition of 1 U RQ1 Dnase. Radioactive RNA was extracted with 100 AL of a HENS buffer containing 20 mM HEPES (pH 7.0), 5 mM EDTA, 50 mM NaCl, 0.1% SDS, and tRNA (1 Ag/AL). This last compound removed unincorporated nucleotides. The RNA was precipitated 30 min at 80 8C by adding 65 AL of 7.5 M ammonium acetate and 500 AL of cold absolute ethanol. After centrifugation (20 min, 15,000 g), the pellet was dried, redissolved in HENS buffer, and then heated at 65 8C for 1 min. This final solution of radioactive RNA was counted. 1.6. Hybridization 1.6.1. Experiment 1 Slides were placed in paraformaldehyde for 15 min on ice, then rinsed two times in PBS for 5 min. They were dipped in 0.25% anhydric acetic acid/0.1 M TEA for 10 min and rinsed in 2 SSC (0.3 M NaCl, 30 mM sodium citrate) for 20 s. Slides were dehydrated with ethanol and successively put in 5 mM MgCl2–PBS for 10 min, in 0.25 M Tris–0.1 M glycine for 10 min, and in 50% formamide-2 SET (0.3 M NaCl, 60 mM Tris–HCl, 4 mM EDTA, pH 8.0) at 37 8C for 10 min. The hybridization mixture consisting of 7.25 mL sterile water, 2.5 mL 20 SET, 0.1 g PVP (0.1 AM), 0.1 g Ficoll (0.01 M), 0.1 g BSA, 12.5 mL formamide (13 M), 4 g dextran sulfate, 50 mg/ mL tRNA, and 500 AL DTT (1 M) was prepared with ~-35S-labeled sense or antisense probes to obtain a concentration of 2.107 cpm/mL. These solutions (sense or antisense) were spotted on slides and a coverslip was placed on the sections. Slides were incubated at 45 8C for 4 h in a humid hybridization chamber. After incubation, the slides were washed rapidly by submersion in 4 SSC, then in 50% formamide-2 SET-0.04% mercaptoethanol for 15 min at 60 8C and lastly in 4 SSC. Slides were incubated in Rnase A solution (10 mg/mL)-3 SET at 37 8C for 20 min and rinsed in 1 SSC at room temperature for 30 min with a light agitation. The last wash consisted of 0.1% mercaptoethanol in 0.2 SET at 50 8C for 30 min. Slides were dehydrated in 30%, 50%, 70% ethanol containing 0.3 M ammonium acetate, and after in 85%, 95%, and 100% ethanol. The hybridized sections were dried and then autoradiographed. Slides were dipped in Hypercoat LM-1 emulsion (Amersham) and exposed for 30 days at 4 8C. At the time of
developing, slides were put in D-19 developer (Kodak) for 2.5 min at 15 8C, rinsed, fixed (fixer Kodak) for 5 min, and placed in water for 10 min. Slides were colored with Harris’ hematoxylin and aqueous eosin. 1.6.2. Experiment 2 Slides were incubated at 45 8C for 6 h instead of 4 h. 1.6.3. Experiment 3 Slides were incubated overnight at 45 8C in a humid hybridization chamber. The succession of wash was different. After incubation, the slides were washed rapidly by submersion in 2 SSC, then incubated for 1 h in 200 mL of 2 SSC with 1 mL of 1 M DTT. Slides were then placed in 50% formamide-2 SSC-10 mM DTT for 20 min at 60 8C and immediately after rinsed in 3 baths of 2 SSC for 3 min each at room temperature. Slides were washed in 2 SSC for 1 h with a light agitation. The last wash consisted on 0.1 SSC for 30 min. The end of the experiment was the same as in experiment 1. 1.7. Exposure time Slides were processed following experiment 1 and then exposed for 20, 30, 40, or 60 days at 4 8C before developing.
2. Results 2.1. cDNA characterization As the probe for the DHAP-AT RNA was not commercially available, it was synthesized and then sequenced for control. The sequence (Fig. 1) of the synthesized cDNA was compared with that of the Rattus norvegicus acyl-CoA DHAP-AT gene (GenBank accession no NM_053410). A total homology was found with a part of the coding sequence between the 1473 and 1877 nucleotides. This synthesized 405-bp probe was used for all the experiments. 2.2. Choice of the optimal parameters for the hybridization (data not shown) Experiments 1 and 2 were tested because the fixation of tissues is often necessary to preserve the cell structure. However, experiment 3, faster, gave satisfactory results and was finally chosen. We considered that a step of fixation was not necessary in our case because we did not study a localization of mRNA at the ultrastructural level but only at the structural level. Moreover, the thinner the slide is, the better the observation will be; the best results were obtained with 8-Am-thickness slides. The composition of the hybridization buffer must provide optimal conditions for the formation of hybrids at the
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Fig. 1. Nucleotide sequence of DHAP-AT probe. A 405-bp PCR product generated from brain RNA with specific primers (underlined) to the rat DHAP-AT was sequenced. The numbers beside the two nucleotides highlighted represent their positions from the beginning of the DHAP-AT gene initially cloned.
incubation temperature. Dextran sulfate was added to accelerate the speed of hybridization process. Ficoll and polyvinylpyrrolidone, high molecular mass molecules, favor the hybridization. BSA decrease electrostatic bonds. tRNA was added in order to reduce non-specific adsorption between the probe and the tissue section. The duration of incubation was also tested (experiment 1 vs. 2 and 3). A long period of incubation was not necessary in our experiments as illustrated by the lack of improvement of the intensity of the signal. Moreover, a short period of incubation is preferable because tissues can be damaged and dried if the humidity of the chamber is not well controlled. The technique developed gave qualitative but not quantitative results; as a consequence, it was not useful to increase the duration of incubation. The time of wash and agitation are two critical parameters because they determine the intensity of the background. Indeed, washes eliminate the aspecific hybrids and also aspecific adsorption between the probe and the different components of the cell. In experiment 1, the total wash time was 95 min whereas it was 180 min in experiment 3. The background was insignificant in the first experiment that’s why we considered that this time of wash was sufficient and it was not necessary to prolong it. For the liver, an exposure of 20 days appeared to be sufficient to reveal a good signal. However, for brain slides, an exposure of 30 days was necessary. Longer exposures were useless. The exposure time must be adapted for each structure because of the level of hybridization. The following results correspond to the experiment made in the optimal conditions.
2.3. In situ hybridization on liver (Fig. 2) The radioactive signal for the sense probe, characterizing the background, was very weak (Fig. 2C). This confirmed the specificity of the antisense probe, the signal being specific of the DHAP-AT mRNA. In the liver (Fig. 2A), the signal of the antisense was very high and homogeneous. 2.4. In situ hybridization on brain (Fig. 3) The radioactive signal of the sense probe was as weak in the brain as in the liver (Figs. 3C1, C2, and C3). In the brain, the signal of the antisense was weaker and different in each structure. In the frontal cortex (Fig. 3A2), the in situ hybridization did not revealed any expression of DHAP-AT. The comparison of some images in contrast phase vs. dark field showed a specific radioactivity localization in the area of the hippocampus (Fig. 3A1) and in the cerebellum (Fig. 3A3). The signal was especially high in the internal structure of the cerebellum, in the hippocampus, and in the corpus callosum.
3. Discussion The aim of this work was to develop in situ hybridization of DHAP-AT mRNA. The liver was chosen because of the high expression and activity of the studied enzyme and allowed to validate the technique. Indeed, the background is very weak and even non-existent, whereas the specific signal of the DHAP-AT mRNA was very high. The optimal
Fig. 2. In situ hybridization of DHAP-AT in the liver. A and B correspond to an observation of the hybridization with the antisense probe in dark field or contrast phase, respectively. C and D correspond to an observation of the hybridization with the sense probe in dark field or contrast phase, respectively.
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Fig. 3. In situ hybridization of DHAP-AT in the brain. 1, 2, and 3 correspond to observations made in the area of the hippocampus, in the cortex and in the cerebellum, respectively. A and B correspond to an observation of the hybridization with the antisense probe in dark field or contrast phase, respectively. C and D correspond to an observation of the hybridization with the sense probe in dark field or contrast phase, respectively. All images were taken with a 100 objective using an Axiovert 25 Zeiss microscope.
conditions determined for in situ hybridization on the liver were a 4-h incubation period and a 95-min wash period. Slides were then exposed for 20 days before developing. The high signal in this tissue may be explained by the high content of peroxisomes, subcellular organelles known to contain the DHAP-AT [11]. Moreover, this high expression is closely related with a high enzymatic activity (data not shown). The liver produces PlsGpl, does not accumulate them (less than 5% of the ethanolamine phospholipids are the plasmalogen form), but secretes nascent lipoproteins in which 20–30% of the ethanolamine phospholipids are plasmenylethanolamine [13]. The PlsGpl synthesis and exportation are both important. Such information may explain the paradox between the high expression of DHAP-AT in the liver and its low level of PlsGpl. This method is of great interest because it can be also transposed on little structures in order to determine a specific localization of the DHAP-AT mRNA and on structures with a light level of expression.
By way of illustration, in situ hybridization was performed on brain with a particular focus on white vs. gray matter and also on some specific structures like cerebellum, hippocampus, and cortex. Indeed, the brain has a complex organization and a particular structure. It is composed of white and gray matters: about 40% of the human brain is made up of gray matter whereas 60% is white matter. The white matter contains lots of nerve fibers that are ensheathed in a membrane called myelin. About 40% of the ethanolamine glycerophospholipids are plasmalogens in human gray matter [5] and 70% in white matter. We can suppose that in a plasmalogen-rich structure, the expression of DHAP-AT gene is intense which leads to a high level of mRNA. The technique used on the liver was applied on the brain with only one little adaptation: the duration of exposure was prolonged to 30 days for optimal results. The expression of DHAP-AT in the brain appeared weak in contrast with its intense expression in the liver. In the liver, a major function of plasmalogen biosynthetic enzymes may be the provision of
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plasmalogen for secretion into lipoproteins. Brain plasmalogens cannot be of liver origin because entire phospholipids cannot go through the hematoencephalic barrier. In the brain, DHAP-AT expression is weaker than in the liver but may be sufficient to synthesize and maintain a plasmalogen pool. We identified in the rat brain a structure–localization relationship. Indeed, our results showed a high signal in the internal structure of the cerebellum, in the hippocampus and in the corpus callosum. This latter structure is a thick band of nerve fibers located between the cerebral hemispheres. It is an oval-shaped center of white substance, surrounded on all sides by a narrow convoluted margin of gray matter, which presents an equal thickness in nearly every part. Fibers of the corpus callosum contain lots of myelinated axons that is why this structure is white. In the same way, the cerebellum contains more white matter than the other areas and it is known that white matter, whose myelin represents the main constituent, is especially rich in PlsGpl. These two marked structures are both rich in myelin and in plasmalogens. Such information may easily explain results obtained by in situ hybridization. Other peroxisomal proteins and their mRNA have been localized in the white matter [6,14]. Such information are in concordance with our results: DHAP-AT is a peroxisomal protein and its mRNA is mainly localized in white matter. Marked structures are rich in myelin. Oligodendrocytes are the myelin-forming cells of the central nervous system [1]. Enzymes for the synthesis of myelin glycerophospholipids are found in the oligodendrocyte cytoplasm that is why we may suppose that DHAP-AT localizes in these cells too. In the frontal cortex, the in situ hybridization did not revealed any expression of DHAP-AT. However, this structure contains plasmalogens [7]. We can hypothesize that the level of DHAP-AT expression is too weak to be detected by the method used. In conclusion, all experiments performed allowed us to determine the optimal conditions to use for in situ hybridization on the brain. This technique is a very powerful technique especially for the specific cellular and/or tissue localization of particular gene expression. Moreover, it is the only technique which allows a visualization in situ of mRNA. It appeared sensitive enough to be used on little structures with a light expression of the studied enzyme. However, a major disadvantage of this technique is the difficulty to assess quantitative differences. For a precise quantification, it is necessary to have recourse to PCR or Northern blotting. DHAP-AT mRNA are localized all over the liver whereas the expression within the brain seems to be specific to structures rich in white matter (the corpus callosum and the internal structure of the cerebellum), which contain plasmalogen-rich myelin. Studies concerning
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the effect of fatty acids on the regulation of the DHAP-AT expression in the brain are in progress.
Acknowledgments The authors gratefully acknowledge MA Maire for her technical assistance. A Tessier is acknowledged for her interest to the study and valuable comments.
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