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Human neuronal calcium sensor-1 shows the highest expression level in cerebral cortex
Neuroscience Letters 319 (2002) 67–70 www.elsevier.com/locate/neulet
Human neuronal calcium sensor-1 shows the highest expression level in cerebral cortex Chiyuan Chen, Long Yu*, Pingzhao Zhang, Jianming Jiang, Yazhou Zhang, Xiaosong Chen, Qi Wu, Qianhong Wu, Shouyuan Zhao State Key Laboratory of Genetics Engineering, Institute of Genetics, School of Life Sciences, Fudan University, No. 220, Handan Road, Shanghai, 200433, P. R. China Received 16 November 2001; accepted 27 November 2001
Abstract Neuronal calcium sensors (NCS) are important constituents in the intracellular signaling pathways. A novel human gene, NCS-1, was identified in the present study. Among the 16 human tissues examined, NCS-1 is expressed most abundantly in the brain. Among the brain regions, the expression level of NCS-1 in cerebral cortex is the highest, which is about six times higher than the average level of the whole brain and a hundred times higher than the spinal cord. In the 12 different anatomical regions of human brain, the expression level of NCS-1 is very high in the temporal lobe, occipital pole, frontal lobe, thalamus, amygdala and hippocampus; moderate in cerebellum, putamen, caudate nucleus; low in the medulla, substantia nigra and the lowest in corpus callosum. Our results suggest that NCS-1 in human brain might be involved in a variety of brain functions such as sensory processing, motor control, emotional control, learning and memory. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neuronal calcium sensors; Gene cloning; Gene expression pattern; Human brain; Cerebral cortex
Calcium regulates a large amount of cellular processes, including cell cycle, cell metabolism and many important signaling transduction pathways. A rapidly growing family of calcium-binding proteins, neuronal calcium sensors (NCS), is involved in the regulation of these processes. To date, more than 50 members of this family have been isolated in various species and, according to their sequence similarity, can be grouped into six subfamilies, i.e. frequenins (NCS-1), recoverins, visinin-like proteins, guanyl cyclase-activating proteins, Ce-NCS-2 [2] and Kv channel-interacting proteins [1]. NCS-1 is an important member of the frequenin subfamily. Previous studies showed that it could facilitate neurotransmission by modulating sodium/ calcium exchange and the K 1-currents [9,10]. NCS-1 was also implicated in the modulation of calcium/calmodulindependent enzymes involved in neuronal signal transduction [11] and probably played an important role in regulating calcium-dependent phosphorylation in nervous system [3]. Genes encoding NCS-1 protein have been isolated in worm, fly, frog, chicken, mouse and rat [2]. NCS-1 is mainly localized in the myelinated axons, the axonal ramifications * Tel.: 186-21-65642422; fax: 186-21-65643250. E-mail address: [email protected] (L. Yu).
of the basket cell in the cerebellar cortex, and the large neurons in the brainstem and pons. The subcellular localization shows that NCS-1 exists preferentially in the perikarya, which are associated with the membranes of the trans saccules of the Golgi apparatus [5]. In chicken, it distributes in cerebellum, forebrain, brain stem and midbrain [6]. In mice, it is ubiquitously expressed in most part of the brain, especially high in the hippocampus [7]. In the human central nervous system, however, the regional distribution of NCS-1 expression has not yet been reported. Molecular cloning and Northern hybridization were carried out in our laboratory to investigate the distribution of NCS-1 in human nervous system, especially in the central nervous system, as well as the physiological role of the NCS-1. The cDNA sequence of the rat NCS-1 (GenBank accession No.L27421) was used to search the human EST division in GenBank. The homologous ESTs obtained were compiled into a contig of about 1.2 kb by the assembly program of Wisconsin Package (Version 10.0, Genetics Computer Group, Madison, WI, USA). According to the contig sequence, two primers, A (5 0 -CCGAGGATGGGGAAATCCAACAG-3 0 , nt 62-84) and B (5 0 -ATCCAGCTCCAGCCTGGGACTAT-3 0 , nt 637-659) were designed and used for PCR amplification from several human cDNA libraries
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 55 5- 1
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C. Chen et al. / Neuroscience Letters 319 (2002) 67–70
Fig. 1. (A) The nucleotide and deduced amino acid sequences of NCS-1. The recoverin homology domain is underlined. The EF-hand motifs are boxed and in italics. The NH2-terminal glycin is highlighted. Three putative polyadenylation signals at the 3 0 UTR are italic and bold. (B) The NCS-1 gene in the BAC clone RP11-88G17 is shown by the horizontal arrow. The positions of BAC short tandem repeat sequences, (CA)17 and (TG)20, are indicated in the BAC. One of the gap in the BAC clone, which is located within NCS-1 and filled by us, is indicated by + . The other gap is indicated by SS. The schematic gene structure of NCS-1 is shown in the bottom. The black rectangles indicate the exons. * indicates start codon. K indicates stop codon.
(Clontech). An expected cDNA fragment of 600 bp was obtained from human brain lgt11 cDNA library and was sequenced by a BigDye terminator sequencing kit with ABI377 sequencer (Perkin-Elmer applied Biosystems, Foster City, CA, USA). The cDNA had a complete open reading frame encoding 190 amino acids (nt 68-637), which was predicted to be a polypeptide of 21.8 KDa. Like other members of frequenin subfamily, NCS-1 also contained 4 EF-hand motifs, a N-terminal recoverin homology domain and a consensus NH2-terminal glycin for Nterminal myristoylation (Fig. 1A). The sequences of amino acid in human, rat, chicken NCS-1 and mouse frequenin were
100% identical. To determine the relationship between NCS1 and the other NCS proteins, a strict consensus neighborjoining tree using a phylogenetic analysis program (Clustal X) was reconstructed (Fig. 2). Forty-eight members of NCS family in different species are divided into six groups, the human NCS-1 being in the fourth group that contains ten homologs. Human NCS-1 cDNA was applied to Northern blot analysis on the multiple-tissue Northern membranes (MTN I and MTN II, Clontech) carrying 16 human tissue mRNAs. A 4.5 kb NCS-1 transcript, which was similar to the 4.2 kb transcript of its ortholog, Mfreq [7], was found with the highest
C. Chen et al. / Neuroscience Letters 319 (2002) 67–70
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from human brain lgt11 cDNA library by PCR. Confirmed by sequencing, the a- 32P-dATP-labeled (Amersham) CD fragment was used to screen human brain lgt11 cDNA library and a full-length NCS-1 cDNA of 4.3 kb (GenBank accession No. AF134479) with two canonical polyadenylation signals at nt 4245-4250 and 4273-4278 was obtained. The cDNA sequence and the 4.5 kb transcript shown in Northern hybridization were nearly the same in length. Another atypical polyadenylation signal, ATTAATTA, was found at nt 1216–1223. The employment of this polyadenylation signal, which could be supported by the ESTs AA599118 and AI479413, would generate a transcript that was similar to the 1.4 kb band found in Northern blot analysis (Fig. 3A). In addition, the chromosomal localization of human NCS1 was determined with the GB4 radiation hybrid cell panel (Research Genetics). The result showed that NCS-1 was linked, with a LOD score of 6.2, to the marker AFM249yh9 which was mapped to 9q34.1. By the BLAST analysis, we noticed that NSC-1 cDNA sequence matches one BAC clone (AL360004, at nt 44027-157, 72388-142, 89262-400, 91155-233, 94080-168, 94512589, 97819-934 and 104420-108054). The result shows that NCS-1 gene spans a region of about 64 kb, containing at least eight exons and seven introns (Fig. 1B). To investigate the possible relationship between the expression level of NCS-1 and some neural physiological functions, the 600 bp cDNA probe mentioned above was
Fig. 2. The phylogenetic tree of the NCS protein family. The evolutionary relationships of these sequences are calculated by Clustal X program. The lengths of horizontal lines are proportional to the estimated genetic distances between the sequences. The first letters denote the species: d, Drosophila; c.eleg., C. elegance; b, bovine; rp, Rana pipiens; rc, Rana catesbeiana; nf, Naegleria fowleri; ng, Naegleria gruberi; fn, Filobasidiella neoformans; r, rat; m, mouse; c, chicken; x, Xenopus; pi, Panulirus interruptus; bt, Bos Taurus; sc, Saccharomyces cerevisiae.
expression level in brain, and moderate expression level in prostate, kidney, small intestine, ovary and colon (Fig. 3A). Although a small transcript of 1.4 kb was also detected in these tissues, its expression level was very low compared with the 4.5 kb transcript. The length of the small transcript was corresponding to the 1.2 Kb EST contig, therefore it would represent one type of NCS-1 transcripts. The 4.5 kb transcript, on the other hand, led to the suspicion of the existence of another transcript which has an unidentified longer 3 0 UTR. To examine our hypothesis, two primers C (5 0 -ATAGTCCCAGGCTGGAGCTGGAT-3 0 , nt 637-659) and D (5 0 TGCGAGGCAGCCAAGTCCTTGGTT-3 0 , nt 908-931) were designed. A unique 300 bp fragment was amplified
Fig. 3. Northern hybridization of human NCS-1 with total mRNA from 16 human tissues (A) and 15 regions of human brain (B) with b -actin as a control. The intensity of the hybridized bands were scanned with GDS-800 (Bio-RAD) and Annutatin Grabber-1 T2.51 scanner software as well as UVP Gelworks ID Advanced Version 2.51 analysis software. The b -actin was scanned similarly. The ratio of expression intensity in 16 tissues and 15 brain regions to those of b -actin in the same tissues or regions were used as the index of expression level in different tissues and regions.
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used to perform Northern blot analysis on human brain MTN II and brain MTN IV (Clontech) carrying mRNA from 12 brain regions, cerebral cortex, spinal cord and the whole brain. Among these neural tissues examined, the expression level of NCS-1 in cerebral cortex is the highest, about six times the average level of the whole brain and a hundred times of the spinal cord. The result in Fig. 3B showed that NCS-1 was abundantly expressed in those brain regions associated with sensory processing, learning, memory and emotional control functions, such as: temporal lobe; occipital pole; thalamus; frontal lobe; hippocampus; and amygdala. NCS-1 was moderately expressed in those regions associated with motor control and automatic activity control functions, such as: cerebellum; putamen; caudate nucleus; substantia nigra; and medulla. Interestingly, the expression level of NCS-1 in cerebral cortex was 2-fold higher than that in temporal lobe, while in spinal cord and corpus callosum it was very low or nearly undetectable. The previous studies of NCS-1 gene in fruit fly, chicken, bovine, rat and mouse had suggested that the expression level of NCS-1 was higher in brain regions or nerve tissues related with sensory processing than in those associated with motor control. Our studies also revealed that NCS-1 was highly expressed as well in those regions associated with emotional control, learning and memory functions, and moderately expressed in regions related with automatic activity control. The expression features of either NCS-1 or frequetin in nervous systems of different species have been reported. NCS-1 is mainly presented in the inner segment of the photoreceptor, the ganglion cells of the retina and the inner plexiform layer in rat, bovine and chick [4]; in central horn spinal cord and hippocampus in mouse [7] and in the larval neuromuscular junctions in Drosophila [8]. Besides, over-expression of frequetin in mature motor neurons in Drosophila leads to facilitation of synaptic responses to high frequency stimulation, while basal synaptic transmission is not affected [8,10]. In addition, in crayfish, a frequetin-like molecule is selectively expressed in phasic but not tonic motoneurons [12].The expression patterns of different human brain regions discovered in the present study would enrich the understanding of both NCS-1 and frequetin genes and their role in intracellular signaling pathways of various neural cells localized at different human brain functional regions. This work was supported by the National 973 Program, 863 High Technology Program (863-Z-02-04-01) and the
National Science Foundation (30024001, 39680019) of China. [1] An, W.F., Bowlby, M.R., Betty, M., Cao, J., Ling, H.P., Mendoza, G., Hinson, J.W., Mattsson, K.I., Strassle, B.W., Trimmer, J.S. and Rhodes, K.J., Modulation of A-type potassium channels by a family of calcium sensors, Nature, 403 (2000) 553–556. [2] Braunewell, K.H. and Gundelfinger, E.D., Intracellular neuronal calcium sensor proteins: a family of EF-hand calcium-binding proteins in search of a function, Cell Tissue Res., 295 (1999) 1–12. [3] De Castro, E., Nef, S., Fiumelli, H., Lenz, S.E., Kawamura, S. and Nef, P., Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors, Biochem. Biophys. Res. Commum., 216 (1995) 133–140. [4] De Raad, S., Comte, M., Nef, P., Lenz, S.E., Gundelfinger, E.D. and Cox, J.A., Distribution pattern of three neural calcium-binding proteins (NCS-1, VILIP and recoverin) in chicken, bovine and rat retina, Histochem. J., 27 (1995) 524–535. [5] Martone, M.E., Edelmann, V.M., Ellisman, M.H. and Nef, P., Cellular and subcellular distribution of the calcium-binding protein NCS-1 in the central nervous system of the rat, Cell Tissue Res., 295 (1999) 395–407. [6] Nef, S., Fiumelli, H., De Castro, E., Raes, M.B. and Nef, P., Identification of neuronal calcium sensor (NCS-1) possibly involved in the regulation of receptor phosphorylation, J. Recept. Signal Transduct. Res., 15 (1995) 365–378. [7] Olafsson, P., Soares, H.D., Herzog, K.H., Wang, T., Morgan, J.I. and Lu, B., The Ca 21 binding protein, frequenin is a nervous system-specific protein in mouse preferentially localized in neuritis, Mol Brain Res, 44 (1997) 73–82. [8] Pongs, O., Lindemeier, J., Zhu, X.R., Theil, T., Engelkamp, D., Krah-Jentgens, I., Lambrecht, H.G., Koch, K.W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J., Canal, I., Barbas, J.A. and Ferrus, A., Frequenin–a novel calciumbinding protein that modulates synaptic efficacy in the Drosophila nervous system, Neuron, 11 (1993) 15–28. [9] Poulain, C., Ferrus, A. and Mallart, A., Modulation of type A K 1 current in Drosophila larval muscle by internal Ca2 1; effects of the overexpression of frequenin, Pflugers Arch., 427 (1994) 71–79. [10] Rivosecchi, R., Pongs, O., Theil, T. and Mallart, A., Implication of frequenin in the facilitation of transmitter release in Drosophila, J. Physiol., 474 (1994) 223–232. [11] Schaad, N.C., De Castro, E., Nef, S., Hegi, S., Hinrichsen, R., Martone, M.E., Ellisman, M.H., Sikkink, R., Rusnak, F., Sygush, J. and Nef, P., Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1, Proc Natl Acad Sci USA, 93 (1996) 9253–9258. [12] Jeromin, A., Shayan, A.J., Msghina, M., Roder, J. and Atwood, H.L., Crustacean frequenins: molecular cloning and differential localization at neuromuscular junctions, J. Neurobiol., (1999) 41165–41175.
Human neuronal calcium sensor-1 shows the highest expression level in cerebral cortex Chiyuan Chen, Long Yu*, Pingzhao Zhang, Jianming Jiang, Yazhou Zhang, Xiaosong Chen, Qi Wu, Qianhong Wu, Shouyuan Zhao State Key Laboratory of Genetics Engineering, Institute of Genetics, School of Life Sciences, Fudan University, No. 220, Handan Road, Shanghai, 200433, P. R. China Received 16 November 2001; accepted 27 November 2001
Abstract Neuronal calcium sensors (NCS) are important constituents in the intracellular signaling pathways. A novel human gene, NCS-1, was identified in the present study. Among the 16 human tissues examined, NCS-1 is expressed most abundantly in the brain. Among the brain regions, the expression level of NCS-1 in cerebral cortex is the highest, which is about six times higher than the average level of the whole brain and a hundred times higher than the spinal cord. In the 12 different anatomical regions of human brain, the expression level of NCS-1 is very high in the temporal lobe, occipital pole, frontal lobe, thalamus, amygdala and hippocampus; moderate in cerebellum, putamen, caudate nucleus; low in the medulla, substantia nigra and the lowest in corpus callosum. Our results suggest that NCS-1 in human brain might be involved in a variety of brain functions such as sensory processing, motor control, emotional control, learning and memory. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neuronal calcium sensors; Gene cloning; Gene expression pattern; Human brain; Cerebral cortex
Calcium regulates a large amount of cellular processes, including cell cycle, cell metabolism and many important signaling transduction pathways. A rapidly growing family of calcium-binding proteins, neuronal calcium sensors (NCS), is involved in the regulation of these processes. To date, more than 50 members of this family have been isolated in various species and, according to their sequence similarity, can be grouped into six subfamilies, i.e. frequenins (NCS-1), recoverins, visinin-like proteins, guanyl cyclase-activating proteins, Ce-NCS-2 [2] and Kv channel-interacting proteins [1]. NCS-1 is an important member of the frequenin subfamily. Previous studies showed that it could facilitate neurotransmission by modulating sodium/ calcium exchange and the K 1-currents [9,10]. NCS-1 was also implicated in the modulation of calcium/calmodulindependent enzymes involved in neuronal signal transduction [11] and probably played an important role in regulating calcium-dependent phosphorylation in nervous system [3]. Genes encoding NCS-1 protein have been isolated in worm, fly, frog, chicken, mouse and rat [2]. NCS-1 is mainly localized in the myelinated axons, the axonal ramifications * Tel.: 186-21-65642422; fax: 186-21-65643250. E-mail address: [email protected] (L. Yu).
of the basket cell in the cerebellar cortex, and the large neurons in the brainstem and pons. The subcellular localization shows that NCS-1 exists preferentially in the perikarya, which are associated with the membranes of the trans saccules of the Golgi apparatus [5]. In chicken, it distributes in cerebellum, forebrain, brain stem and midbrain [6]. In mice, it is ubiquitously expressed in most part of the brain, especially high in the hippocampus [7]. In the human central nervous system, however, the regional distribution of NCS-1 expression has not yet been reported. Molecular cloning and Northern hybridization were carried out in our laboratory to investigate the distribution of NCS-1 in human nervous system, especially in the central nervous system, as well as the physiological role of the NCS-1. The cDNA sequence of the rat NCS-1 (GenBank accession No.L27421) was used to search the human EST division in GenBank. The homologous ESTs obtained were compiled into a contig of about 1.2 kb by the assembly program of Wisconsin Package (Version 10.0, Genetics Computer Group, Madison, WI, USA). According to the contig sequence, two primers, A (5 0 -CCGAGGATGGGGAAATCCAACAG-3 0 , nt 62-84) and B (5 0 -ATCCAGCTCCAGCCTGGGACTAT-3 0 , nt 637-659) were designed and used for PCR amplification from several human cDNA libraries
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 55 5- 1
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C. Chen et al. / Neuroscience Letters 319 (2002) 67–70
Fig. 1. (A) The nucleotide and deduced amino acid sequences of NCS-1. The recoverin homology domain is underlined. The EF-hand motifs are boxed and in italics. The NH2-terminal glycin is highlighted. Three putative polyadenylation signals at the 3 0 UTR are italic and bold. (B) The NCS-1 gene in the BAC clone RP11-88G17 is shown by the horizontal arrow. The positions of BAC short tandem repeat sequences, (CA)17 and (TG)20, are indicated in the BAC. One of the gap in the BAC clone, which is located within NCS-1 and filled by us, is indicated by + . The other gap is indicated by SS. The schematic gene structure of NCS-1 is shown in the bottom. The black rectangles indicate the exons. * indicates start codon. K indicates stop codon.
(Clontech). An expected cDNA fragment of 600 bp was obtained from human brain lgt11 cDNA library and was sequenced by a BigDye terminator sequencing kit with ABI377 sequencer (Perkin-Elmer applied Biosystems, Foster City, CA, USA). The cDNA had a complete open reading frame encoding 190 amino acids (nt 68-637), which was predicted to be a polypeptide of 21.8 KDa. Like other members of frequenin subfamily, NCS-1 also contained 4 EF-hand motifs, a N-terminal recoverin homology domain and a consensus NH2-terminal glycin for Nterminal myristoylation (Fig. 1A). The sequences of amino acid in human, rat, chicken NCS-1 and mouse frequenin were
100% identical. To determine the relationship between NCS1 and the other NCS proteins, a strict consensus neighborjoining tree using a phylogenetic analysis program (Clustal X) was reconstructed (Fig. 2). Forty-eight members of NCS family in different species are divided into six groups, the human NCS-1 being in the fourth group that contains ten homologs. Human NCS-1 cDNA was applied to Northern blot analysis on the multiple-tissue Northern membranes (MTN I and MTN II, Clontech) carrying 16 human tissue mRNAs. A 4.5 kb NCS-1 transcript, which was similar to the 4.2 kb transcript of its ortholog, Mfreq [7], was found with the highest
C. Chen et al. / Neuroscience Letters 319 (2002) 67–70
69
from human brain lgt11 cDNA library by PCR. Confirmed by sequencing, the a- 32P-dATP-labeled (Amersham) CD fragment was used to screen human brain lgt11 cDNA library and a full-length NCS-1 cDNA of 4.3 kb (GenBank accession No. AF134479) with two canonical polyadenylation signals at nt 4245-4250 and 4273-4278 was obtained. The cDNA sequence and the 4.5 kb transcript shown in Northern hybridization were nearly the same in length. Another atypical polyadenylation signal, ATTAATTA, was found at nt 1216–1223. The employment of this polyadenylation signal, which could be supported by the ESTs AA599118 and AI479413, would generate a transcript that was similar to the 1.4 kb band found in Northern blot analysis (Fig. 3A). In addition, the chromosomal localization of human NCS1 was determined with the GB4 radiation hybrid cell panel (Research Genetics). The result showed that NCS-1 was linked, with a LOD score of 6.2, to the marker AFM249yh9 which was mapped to 9q34.1. By the BLAST analysis, we noticed that NSC-1 cDNA sequence matches one BAC clone (AL360004, at nt 44027-157, 72388-142, 89262-400, 91155-233, 94080-168, 94512589, 97819-934 and 104420-108054). The result shows that NCS-1 gene spans a region of about 64 kb, containing at least eight exons and seven introns (Fig. 1B). To investigate the possible relationship between the expression level of NCS-1 and some neural physiological functions, the 600 bp cDNA probe mentioned above was
Fig. 2. The phylogenetic tree of the NCS protein family. The evolutionary relationships of these sequences are calculated by Clustal X program. The lengths of horizontal lines are proportional to the estimated genetic distances between the sequences. The first letters denote the species: d, Drosophila; c.eleg., C. elegance; b, bovine; rp, Rana pipiens; rc, Rana catesbeiana; nf, Naegleria fowleri; ng, Naegleria gruberi; fn, Filobasidiella neoformans; r, rat; m, mouse; c, chicken; x, Xenopus; pi, Panulirus interruptus; bt, Bos Taurus; sc, Saccharomyces cerevisiae.
expression level in brain, and moderate expression level in prostate, kidney, small intestine, ovary and colon (Fig. 3A). Although a small transcript of 1.4 kb was also detected in these tissues, its expression level was very low compared with the 4.5 kb transcript. The length of the small transcript was corresponding to the 1.2 Kb EST contig, therefore it would represent one type of NCS-1 transcripts. The 4.5 kb transcript, on the other hand, led to the suspicion of the existence of another transcript which has an unidentified longer 3 0 UTR. To examine our hypothesis, two primers C (5 0 -ATAGTCCCAGGCTGGAGCTGGAT-3 0 , nt 637-659) and D (5 0 TGCGAGGCAGCCAAGTCCTTGGTT-3 0 , nt 908-931) were designed. A unique 300 bp fragment was amplified
Fig. 3. Northern hybridization of human NCS-1 with total mRNA from 16 human tissues (A) and 15 regions of human brain (B) with b -actin as a control. The intensity of the hybridized bands were scanned with GDS-800 (Bio-RAD) and Annutatin Grabber-1 T2.51 scanner software as well as UVP Gelworks ID Advanced Version 2.51 analysis software. The b -actin was scanned similarly. The ratio of expression intensity in 16 tissues and 15 brain regions to those of b -actin in the same tissues or regions were used as the index of expression level in different tissues and regions.
70
C. Chen et al. / Neuroscience Letters 319 (2002) 67–70
used to perform Northern blot analysis on human brain MTN II and brain MTN IV (Clontech) carrying mRNA from 12 brain regions, cerebral cortex, spinal cord and the whole brain. Among these neural tissues examined, the expression level of NCS-1 in cerebral cortex is the highest, about six times the average level of the whole brain and a hundred times of the spinal cord. The result in Fig. 3B showed that NCS-1 was abundantly expressed in those brain regions associated with sensory processing, learning, memory and emotional control functions, such as: temporal lobe; occipital pole; thalamus; frontal lobe; hippocampus; and amygdala. NCS-1 was moderately expressed in those regions associated with motor control and automatic activity control functions, such as: cerebellum; putamen; caudate nucleus; substantia nigra; and medulla. Interestingly, the expression level of NCS-1 in cerebral cortex was 2-fold higher than that in temporal lobe, while in spinal cord and corpus callosum it was very low or nearly undetectable. The previous studies of NCS-1 gene in fruit fly, chicken, bovine, rat and mouse had suggested that the expression level of NCS-1 was higher in brain regions or nerve tissues related with sensory processing than in those associated with motor control. Our studies also revealed that NCS-1 was highly expressed as well in those regions associated with emotional control, learning and memory functions, and moderately expressed in regions related with automatic activity control. The expression features of either NCS-1 or frequetin in nervous systems of different species have been reported. NCS-1 is mainly presented in the inner segment of the photoreceptor, the ganglion cells of the retina and the inner plexiform layer in rat, bovine and chick [4]; in central horn spinal cord and hippocampus in mouse [7] and in the larval neuromuscular junctions in Drosophila [8]. Besides, over-expression of frequetin in mature motor neurons in Drosophila leads to facilitation of synaptic responses to high frequency stimulation, while basal synaptic transmission is not affected [8,10]. In addition, in crayfish, a frequetin-like molecule is selectively expressed in phasic but not tonic motoneurons [12].The expression patterns of different human brain regions discovered in the present study would enrich the understanding of both NCS-1 and frequetin genes and their role in intracellular signaling pathways of various neural cells localized at different human brain functional regions. This work was supported by the National 973 Program, 863 High Technology Program (863-Z-02-04-01) and the
National Science Foundation (30024001, 39680019) of China. [1] An, W.F., Bowlby, M.R., Betty, M., Cao, J., Ling, H.P., Mendoza, G., Hinson, J.W., Mattsson, K.I., Strassle, B.W., Trimmer, J.S. and Rhodes, K.J., Modulation of A-type potassium channels by a family of calcium sensors, Nature, 403 (2000) 553–556. [2] Braunewell, K.H. and Gundelfinger, E.D., Intracellular neuronal calcium sensor proteins: a family of EF-hand calcium-binding proteins in search of a function, Cell Tissue Res., 295 (1999) 1–12. [3] De Castro, E., Nef, S., Fiumelli, H., Lenz, S.E., Kawamura, S. and Nef, P., Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors, Biochem. Biophys. Res. Commum., 216 (1995) 133–140. [4] De Raad, S., Comte, M., Nef, P., Lenz, S.E., Gundelfinger, E.D. and Cox, J.A., Distribution pattern of three neural calcium-binding proteins (NCS-1, VILIP and recoverin) in chicken, bovine and rat retina, Histochem. J., 27 (1995) 524–535. [5] Martone, M.E., Edelmann, V.M., Ellisman, M.H. and Nef, P., Cellular and subcellular distribution of the calcium-binding protein NCS-1 in the central nervous system of the rat, Cell Tissue Res., 295 (1999) 395–407. [6] Nef, S., Fiumelli, H., De Castro, E., Raes, M.B. and Nef, P., Identification of neuronal calcium sensor (NCS-1) possibly involved in the regulation of receptor phosphorylation, J. Recept. Signal Transduct. Res., 15 (1995) 365–378. [7] Olafsson, P., Soares, H.D., Herzog, K.H., Wang, T., Morgan, J.I. and Lu, B., The Ca 21 binding protein, frequenin is a nervous system-specific protein in mouse preferentially localized in neuritis, Mol Brain Res, 44 (1997) 73–82. [8] Pongs, O., Lindemeier, J., Zhu, X.R., Theil, T., Engelkamp, D., Krah-Jentgens, I., Lambrecht, H.G., Koch, K.W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J., Canal, I., Barbas, J.A. and Ferrus, A., Frequenin–a novel calciumbinding protein that modulates synaptic efficacy in the Drosophila nervous system, Neuron, 11 (1993) 15–28. [9] Poulain, C., Ferrus, A. and Mallart, A., Modulation of type A K 1 current in Drosophila larval muscle by internal Ca2 1; effects of the overexpression of frequenin, Pflugers Arch., 427 (1994) 71–79. [10] Rivosecchi, R., Pongs, O., Theil, T. and Mallart, A., Implication of frequenin in the facilitation of transmitter release in Drosophila, J. Physiol., 474 (1994) 223–232. [11] Schaad, N.C., De Castro, E., Nef, S., Hegi, S., Hinrichsen, R., Martone, M.E., Ellisman, M.H., Sikkink, R., Rusnak, F., Sygush, J. and Nef, P., Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1, Proc Natl Acad Sci USA, 93 (1996) 9253–9258. [12] Jeromin, A., Shayan, A.J., Msghina, M., Roder, J. and Atwood, H.L., Crustacean frequenins: molecular cloning and differential localization at neuromuscular junctions, J. Neurobiol., (1999) 41165–41175.