Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

BMC Neuroscience

Tetsuo Sugimoto¹  Masato Maruyama¹  Masahiko Kase¹  Yuji Yamashita¹  Stefan Trifonov¹ 

[1] Department of Anatomy and Brain Science, Kansai Medical University, Hirakata, Osaka 573-1010, Japan

关键词: in situ hybridization;    Quantitative RT-PCR;    Alternative splicing;    GAD2;    GAD1;   

Others  :  1090902
DOI  :  10.1186/1471-2202-15-114

 received in 2014-07-10, accepted in 2014-09-25,  发布年份 2014

【 摘 要 】

Background

GABA has important functions in brain plasticity related processes like memory, learning, locomotion and during the development of the nervous system. It is synthesized by the glutamic acid decarboxylase (GAD). There are two isoforms of GAD, GAD1 and GAD2, which are encoded by different genes. During embryonic development the transcription of GAD1 mRNA is regulated by alternative splicing and several alternative transcripts were distinguished in human, mouse and rat. Despite the fact that the structure of GAD1 gene has been extensively studied, knowledge of its exact structural organization, alternative promoter usage and splicing have remained incomplete.

Results

In the present study we report the identification and characterization of novel GAD1 splicing isoforms (GenBank: KM102984, KM102985) by analyzing genomic and mRNA sequence data using bioinformatics, cloning and sequencing. Ten mRNA isoforms are generated from GAD1 gene locus by the combined actions of utilizing different promoters and alternative splicing of the coding exons. Using RT-PCR we found that GAD1 isoforms share similar pattern of expression in different mouse tissues and are expressed early during development. Quantitative RT-PCR was used to investigate the expression of GAD1 isoforms and GAD2 in olfactory bulb, cortex, medial and lateral striatum, hippocampus and cerebellum of adult mouse. Olfactory bulb showed the highest expression of GAD1 transcripts. Isoforms 1/2 are the most abundant forms. Their expression is significantly higher in the lateral compared to the medial striatum. Isoforms 3/4, 5/6, 7/8 and 9/10 are barely detectable in all investigated regions except of the high expression in olfactory bulb. When comparing GAD1 expression with GAD2 we found that Isoforms 1/2 are the predominant isoforms. In situ hybridization confirmed the predominant expression of Isoforms 7/8 and 9/10 in the olfactory bulb and revealed their weak expression in hippocampus, cerebellum and some other areas known to express GAD1.

Conclusions

Generation of ten splicing isoforms of GAD1 was described including two so far uncharacterized transcripts. GAD1 splicing isoforms producing the shorter, enzymatically inactive GAD25 protein are expressed at very low level in adult mouse brain except in the olfactory bulb that is associated with neurogenesis and synaptic plasticity even during adulthood.

【 授权许可】

   
2014 Trifonov et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Varju P, Katarova Z, Madarasz E, Szabo G: GABA signalling during development: new data and old questions. Cell Tissue Res 2001, 305:239-246.
• [2]Represa A, Ben-Ari Y: Trophic actions of GABA on neuronal development. Trends Neurosci 2005, 28:278-283.
• [3]Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, Tobin AJ: Two genes encode distinct glutamate decarboxylases. Neuron 1991, 7:91-100.
• [4]Kaufman DL, Houser CR, Tobin AJ: Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. J Neurochem 1991, 56:720-723.
• [5]Soghomonian JJ, Martin DL: Two isoforms of glutamate decarboxylase: why? Trends Pharmacol Sci 1998, 19:500-505.
• [6]Esclapez M, Tillakaratne NJ, Tobin AJ, Houser CR: Comparative localization of mRNAs encoding two forms of glutamic acid decarboxylase with nonradioactive in situ hybridization methods. J Comp Neurol 1993, 331:339-362.
• [7]Esclapez M, Tillakaratne NJ, Kaufman DL, Tobin AJ, Houser CR: Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J Neurosci 1994, 14:1834-1855.
• [8]Feldblum S, Erlander MG, Tobin AJ: Different distributions of GAD65 and GAD67 mRNAs suggest that the two glutamate decarboxylases play distinctive functional roles. J Neurosci Res 1993, 34:689-706.
• [9]Trifonov S, Houtani T, Kase M, Toida K, Maruyama M, Yamashita Y, Shimizu J-I, Sugimoto T: Lateral regions of the rodent striatum reveal elevated glutamate decarboxylase 1 mRNA expression in medium-sized projection neurons. Eur J Neurosci 2012, 35:711-722.
• [10]Martin DL, Martin SB, Wu SJ, Espina N: Regulatory properties of brain glutamate decarboxylase (GAD): the apoenzyme of GAD is present principally as the smaller of two molecular forms of GAD in brain. J Neurosci 1991, 11:2725-2731.
• [11]Battaglioli G, Liu H, Martin DL: Kinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis. J Neurochem 2003, 86:879-887.
• [12]Szabó G, Katarova Z, Körtvély E, Greenspan RJ, Urbán Z: Structure and the promoter region of the mouse gene encoding the 67-kD form of glutamic acid decarboxylase. DNA Cell Biol 1996, 15:1081-1091.
• [13]Yanagawa Y, Kobayashi T, Kamei T, Ishii K, Nishijima M, Takaku A, Tamura S: Structure and alternative promoters of the mouse glutamic acid decarboxylase 67 gene. Biochem J 1997, 326:573-578.
• [14]Bu DF, Tobin AJ: The exon-intron organization of the genes (GAD1 and GAD2) encoding two human glutamate decarboxylases (GAD67 and GAD65) suggests that they derive from a common ancestral GAD. Genomics 1994, 21:222-228.
• [15]Matsukawa S, Ueno H: Analysis of intron-exon positioning on glutamate decarboxylase and its relation with evolution. J Biol Macromol 2007, 7:35-48.
• [16]Küppers E, Sabolek M, Anders U, Pilgrim C, Beyer C: Developmental regulation of glutamic acid decarboxylase mRNA expression and splicing in the rat striatum by dopamine. Mol Brain Res 2000, 81:19-28.
• [17]Hyde TM, Lipska BK, Ali T, Mathew SV, Law AJ, Metitiri OE, Straub RE, Ye T, Colantuoni C, Herman MM, Bigelow LB, Weinberger DR, Kleinman JE: Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia. J Neurosci 2011, 31:11088-11095.
• [18]Krizbai IA, Katarova Z, Szabó G, Párducz A, Wolff JR: Modulation of the truncated GAD25 by estrogen in the olfactory bulb of adult rats. Neuroreport 2000, 11:791-794.
• [19]Szabo G, Katarova Z, Greenspan R: Distinct protein forms are produced from alternatively spliced bicistronic glutamic acid decarboxylase mRNAs during development. Mol Cell Biol 1994, 14:7535-7545.
• [20]Bond RW, Wyborski RJ, Gottlieb DI: Developmentally regulated expression of an exon containing a stop codon in the gene for glutamic acid decarboxylase. Proc Natl Acad Sci U S A 1990, 87:8771-8775.
• [21]Behar T, Ma W, Hudson L, Barker JL: Analysis of the anatomical distribution of GAD67 mRNA encoding truncated glutamic acid decarboxylase proteins in the embryonic rat brain. Dev Brain Res 1994, 77:77-87.
• [22]Chessler SD, Lernmark AA: Alternative splicing of GAD67 results in the synthesis of a third form of glutamic-acid decarboxylase in human islets and other non-neural tissues. J Biol Chem 2000, 275:5188-5192.
• [23]Korpershoek E, Verwest AM, Ijzendoorn Y, Rottier R, Drexhage HA, Krijger RR: Expression of GAD67 and novel GAD67 splice variants during human fetal pancreas development. Endocr Pathol 2007, 18:31-36.
• [24]Liu H, Wang Z, Li S, Zhang Y, Yan Y-C, Li Y: Utilization of an intron located polyadenlyation site resulted in four novel glutamate decarboxylase transcripts. Mol Biol Rep 2009, 36:1469-1474.
• [25]Liu H, Zhang Y, Li S, Yan Y, Li Y: Dynamic regulation of glutamate decarboxylase 67 gene expression by alternative promoters and splicing during rat testis maturation. Mol Biol Rep 2010, 37:3111-3119.
• [26]Trifonov S, Houtani T, Shimizu J, Hamada S, Kase M, Maruyama M, Sugimoto T: GPR155: gene organization, multiple mRNA splice variants and expression in mouse central nervous system. Biochem Biophys Res Commun 2010, 398:19-25.
• [27]Trifonov S, Houtani T, Hamada S, Kase M, Maruyama M, Sugimoto T: In situ hybridization study of the distribution of choline acetyltransferase mRNA and its splice variants in the mouse brain and spinal cord. Neuroscience 2009, 159:344-357.
• [28]Paxinos G, Franklin KBJ: The Mouse Brain in Stereotaxic Coordinates. 2nd edition. San Diego: Academic; 2001.
• [29]Too H-P: Real time PCR quantification of GFRalpha-2 alternatively spliced isoforms in murine brain and peripheral tissues. Brain Res Mol Brain Res 2003, 114:146-153.
• [30]Wan G, Too H-P: Discrimination of alternative spliced isoforms by real-time PCR using locked nucleic acid (LNA) substituted primer. Mol Eng Biol Chem Syst (MEBCS) 2006. http://dspace.mit.edu/handle/1721.1/30385 webcite
• [31]Houtani T, Munemoto Y, Kase M, Sakuma S, Tsutsumi T, Sugimoto T: Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system. Biochem Biophys Res Commun 2005, 335:277-285.
• [32]Chessler SD, Hampe CS, Örtqvist E, Simonson WT, Bekris L: Immune reactivity to GAD25 in type 1 diabetes mellitus. Autoimmunity 2002, 35:335-341.
• [33]Szabo G, Kartarova Z, Hoertnagl B, Somogyi R, Sperk G: Differential regulation of adult and embryonic glutamate decarboxylases in rat dentate granule cells after kainate-induced limbic seizures. Neuroscience 2000, 100:287-295.
• [34]Tillakaratne NJ, Medina-Kauwe L, Gibson KM: Gamma-aminobutyric acid (GABA) metabolism in mammalian neural and nonneural tissues. Comp Biochem Physiol A Physiol 1995, 112:247-263.
• [35]Tillakaratne NJ, Erlander MG, Collard MW, Greif KF, Tobin AJ: Glutamate decarboxylases in nonneural cells of rat testis and oviduct: differential expression of GAD65 and GAD67. J Neurochem 1992, 58:618-627.
• [36]Kim J, Richter W, Aanstoot H-J, Shi Y, Fu Q, Rajotte R, Warnock G, Baekkeskov S: Differential expression of GAD65 and GAD67 in human, rat, and mouse pancreatic islets. Diabetes 1993, 42:1799-1808.
• [37]Petersen JS, Russel S, Marshall MO, Kofod H, Buschard K, Cambon N, Karlsen AE, Boel E, Hagopian WA, Hejnaes KR, Lernmark A, Madsen OD, Michelsen BK: Differential expression of glutamic acid decarboxylase in rat and human islets. Diabetes 1993, 42:484-495.
• [38]Li L, Jiang J, Hagopian WA, Karlsen AE, Skelly M, Baskin DG, Lernmark A: Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies. J Histochem Cytochem 1995, 43:53-59.
• [39]Popp A, Urbach A, Witte OW, Frahm C: Adult and embryonic GAD transcripts are spatiotemporally regulated during postnatal development in the rat brain. PLoS ONE 2009, 4:e4371.
• [40]Schwarzer C, Sperk G: Hippocampal granule cells express glutamic acid decarboxylase-67 after limbic seizures in the rat. Neuroscience 1995, 69:705-709.
• [41]Sloviter RS, Dichter MA, Rachinsky TL, Dean E, Goodman JH, Sollas AL, Martin DL: Basal expression and induction of glutamate decarboxylase and GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus. J Comp Neurol 1996, 373:593-618.