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Genomic Structure and Chromosomal Localization of the Mouse Persyn Gene
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SHORT COMMUNICATION Genomic Structure and Chromosomal Localization of the Mouse Persyn Gene Maria V. Alimova-Kost,* ,† Natalia N. Ninkina,‡ ,§ Stefan Imreh,* Nikolai V. Gnuchev,‡ Jimi Adu,§ Alun M. Davies,§ ,¶ and Vladimir L. Buchman† ,§ ,1 *Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Box 280, S-17177, Stockholm, Sweden; †Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, 117984, Moscow, Russia; ‡Institute of Gene Biology, Russian Academy of Sciences, 34/6, Vavilov Street, Moscow B-334, Russia; §School of Biomedical Sciences, University of St. Andrews, Bute Medical Buildings, St. Andrews, Fife, KY16 9TS, Scotland, United Kingdom; and ¶Neuropa Ltd., Robertson Building, Dumbarton Road, Glasgow G11 6NU, Scotland, United Kingdom Received July 30, 1998; accepted November 17, 1998
Synucleins are a family of small intracellular proteins expressed mainly in the nervous system. The involvement of synucleins in neurodegeneration and malignancy has been demonstrated, but the physiological functions of these proteins remain elusive. Further studies including generation of animals with modified persyn expression are necessary to clarify the functions of these proteins and the mechanisms of their involvement in human diseases. We cloned and determined the organization and chromosomal localization of the mouse gene coding for persyn, a member of the synuclein family. The gene is composed of five exons, and its general structure is very similar to that of the human persyn gene. Using fluorescence in situ hybridization, we assigned the persyn gene to the boundary of bands B and C on mouse chromosome 14. We found a fragment of the gene that directs expression of the persyn protein in sensory neurons and could be used for generation of transgenic animals. © 1999 Academic Press
Persyn is the most recently characterized member of the synuclein family that also includes a-synuclein/ NACP and b-synuclein/PNP 14 (for review see 1, 5). a-synuclein attracted intent attention after a recent demonstration of its direct involvement in the etiology and pathogenesis of serious human neurodegenerative conditions such as Parkinson and Alzheimer diseases (for review see 1, 4, 5, 8, 15, 18). All three members of the synuclein family are found predominantly in neurons but their spatial distribution in the nervous system and developmental time course of expression are Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. AF017255, AF099984, AF099985, and AF099986. 1 To whom correspondence should be addressed. Telephone: 441334-463282. Fax: 44-1334-463600. E-mail: [email protected].
Genomics 56, 224 –227 (1999) Article ID geno.1998.5674 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
very different. a- and b-synucleins are expressed in the central nervous system, predominantly in the brain (10, 12, 13, 17, 19), but persyn is expressed predominantly in the peripheral nervous system and in motoneurons of the spinal cord and motor ganglia (2, 3). Onsets of a- and b-synuclein expression in brain coincide with the end of the embryonic period of development (9, 17); the levels of expression gradually increase during postnatal development and decrease in aging brains (9, 16, 17). In contrast, persyn expression in peripheral sensory and central motoneurons becomes prominent from the very early stages of their development, gradually increasing during the midgestation period, and is steadily high in adults (2, 3). Persyn expression becomes detectable in the cerebral cortex only in adults and, in contrast to a-synuclein, increases with age (2, 16). In addition, a very high level of persyn expression was detected in malignant breast tumors but not in normal breast tissue or benign breast tumors (11, 14). The mechanisms that regulate the spatial and temporal patterns of expression of synucleins are not known. However, understanding these mechanisms is very important for clarifying the biological functions of these proteins and their involvement in human diseases. Of no less importance will be generation of mice with targeted disruption of synuclein genes. The essential step in this direction is to reveal the organization of mouse genes coding for synucleins. We cloned the persyn gene from a mouse genomic library (Stratagene). Four overlapping l-clones were isolated by hybridization with a mouse persyn cDNA clone (3) and mapped using variety of restriction endonucleases (Fig. 1a). The positions of five exons of the gene were determined by a combination of hybridization and PCR approaches. All exons and adjacent regions of introns were sequenced, and no differences between cDNA and exon sequences were found. The general organization, positions of exon–intron bound-
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FIG. 1. (a) Physical map of the mouse persyn genomic locus. (Top) Positions of l clones isolated from a genomic library on the physical map of persyn locus. (Bottom) Detailed map of the mouse persyn gene and a KpnI–BamHI construct used for microinjections and transfections (see text). Positions of exons (black boxes) and the initiation ATG codon (arrow) are shown. (b– d) Localization of persyn gene on mouse metaphase chromosomes using fluorescence in situ hybridization. (b) Metaphase plate; arrows show specific hybridization signals on chromosome 14 obtained with the mixture of biotin-labeled probes lPS11 and lPS91. (c) The same metaphase plate, inverted DAPI banding (black arrows show borders of DAPI bands B and C that hybridized with the probe). (d) Magnified images of chromosome 14 from different metaphase plates. Two images are shown for each chromosome—the FISH signal and inverted DAPI banding.
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aries, and even sizes of introns of the mouse persyn gene are very similar to those of the human persyn gene (14). We used biotin-labeled DNA of clones lPS11 and lPS91 as probes for FISH localization of the persyn gene on mouse metaphase chromosomes. The gene was assigned to the boundary of bands B and C on mouse chromosome 14 (Figs. 1b–1d). This region has syntenic relationships with the 10q23.2– q23.3 region of the human genome where we have localized the human persyn gene (14) as there are a few other known genes (for instance glutamate dehydrogenase gene) localized to these regions of human chromosome 10 and mouse chromosome 14. Information regarding the organization of the mouse persyn gene is very important for further studies of persyn function, particularly for generation of knockout mice. Another powerful approach for studying persyn function is the generation of transgenic mice with increased expression of persyn in cells that normally express this protein. This is especially interesting in light of our recent data about disruption of the neurofilament network in cultured sensory neurons overexpressing persyn (2). The same effect of persyn overexpression in sensory and motoneurons of transgenic animals will suggest the possible involvement of this protein in human neurodegenerative diseases such as giant axonal neuropathy and amyotrophic lateral sclerosis, conditions that severely affect neuronal neurofilaments (6, 7). The best strategy to achieve the right developmental- and cell-specific expression of the protein in transgenic animals is to use the natural regulatory sequences of the gene. To check whether or not a fragment of the mouse persyn gene will direct persyn protein expression in neurons, we constructed a plasmid that contained the whole mouse persyn gene including fragments of upstream and downstream regions but without the internal part (between the NcoI and the XbaI sites) of long intron 3 (see Fig. 1a). This plasmid was either microinjected into cultured mouse sensory neurons or transfected into mouse 3T3 fibroblasts. Forty-eight hours later, cells were fixed and stained with anti-persyn antibody (2, 3). Most of the injected neurons demonstrated intensive staining (Fig. 2a), but no stained fibroblasts were detected, although in parallel cultures transfected with mouse persyn cDNA under Mo-MSV LTR promoter, a substantial percentage of cells expressed persyn protein (Fig. 2b). This result suggests that the region of the mouse persyn gene used in these experiments is sufficient to direct expression of the encoded protein in neurons but not in fibroblasts. Thus, this construct or a similar construct could be used for generation of transgenic mice overexpressing persyn in sensory neurons. In conclusion, we determined the organization and mapped the position of the mouse persyn gene on mouse chromosome 14. These results are important for further functional studies of persyn and other members of the synuclein family.
FIG. 2. (a) Micrograph of mouse sensory (P1 trigeminal ganglia) neurons stained with anti-mouse persyn rabbit polyclonal antibody SK-23 and FITC-labeled secondary antibody. Neurons were microinjected with either a mouse persyn gene plasmid described in the text (above the white line) or a vector plasmid (below the white line) and fixed 48 h later. (b) Micrograph of mouse 3T3 fibroblasts stained with anti-mouse persyn rabbit polyclonal antibody SK-23 and FITClabeled secondary antibody. Fibroblasts were transfected with a plasmid containing mouse persyn cDNA under the control of the Mo-MSV LTR promoter and fixed 48 h later. No stained fibroblasts were seen in parallel cultures transfected with a mouse persyn gene plasmid that directed persyn synthesis in sensory neurons, as illustrated in (a).
ACKNOWLEDGMENTS Our thanks go to Liz Delaney for technical assistance. This work was supported by grants from The Wellcome Trust, The Royal Society, and the Russian Government.
REFERENCES 1.
Buchman, V. L. (1998). Synucleins: Clues to mechanisms of neurodegenerative diseases? Mol. Biol. 32: 592–597. 2. Buchman, V. L., Adu, J., Pino˜n, L. G. P., Ninkina, N. N., and Davies, A. M. (1998). Persyn, a member of the synuclein family, influences neurofilament network integrity. Nature Neurosci. 1: 101–103. 3. Buchman, V. L., Hunter, H. J. A., Pino˜n, L. G. P., Thompson, J., Privalova, E. M., Ninkina, N. N., Lowe, J., and Davies, A. M. (1998). Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system. J. Neurosci. 18: 9335–9341. 4. Chase, T. N. (1997). A gene for Parkinson disease. Arch. Neurol. 54: 1156 –1157.
SHORT COMMUNICATION 5.
6. 7.
8. 9.
10.
11.
12.
13.
Clayton, D. F., and George, J. M. (1998). The synucleins: A family of proteins involved in synaptic function, plasticity, neurodegeneration and disease. Trends Neurosci. 21: 249 –254. Cleveland, D. W. (1996). Neuronal growth and death: Order and disorder in the axoplasm. Cell 84: 663– 666. Donaghy, M., King, R. H., Thomas, P. K., and Workman, J. M. (1988). Abnormalities of the axonal cytoskeleton in giant axonal neuropathy. J. Neurocytol. 17: 197–208. Heintz, N., and Zoghbi, H. (1997). a-synuclein—A link between Parkinson and Alzheimer diseases? Nat. Genet. 16: 325–327. Hsu, L. J., Mallory, M., Xia, Y., Veinbergs, I., Hashimoto, M., Yoshimoto, M., Thal, L. J., Saitoh, T., and Masliah, E. (1998). Expression pattern of synucleins (non-Ab component of Alzheimer’s disease amyloid precursor protein/a-synuclein) during murine brain development. J. Neurochem. 71: 338 – 344. Jakes, R., Spillantini, M. G., and Goedert, M. (1994). Identification of two distinct synucleins from human brain. FEBS Lett. 345: 27–32. Ji, H., Liu, Y. E., Jia, T., Wang, M., Liu, J., Xiao, G., Joseph, B. K., Rosen, C., and Shi, Y. E. (1997). Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing. Cancer Res. 57: 759 –764. Maroteaux, L., and Scheller, R. H. (1991). The rat brain synucleins: Family of proteins transiently associated with neuronal membrane. Mol. Brain Res. 11: 335–343. Nakajo, S., Shioda, S., Nakai, Y., and Nakaya, K. (1994). Localization of phosphoneuroprotein 14 (PNP 14) and its mRNA
14.
15. 16.
17.
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expression in rat brain determined by immunocytochemistry and in situ hybridization. Mol. Brain Res. 27: 81– 86. Ninkina, N., Alimova-Kost, M. V., Paterson, J. W. E., Delaney, L., Cohen, B. B., Imreh, S., Gnuchev, N. V., Davies, A. M., and Buchman, V. L. (1998). Organisation, expression and polymorphism of the human persyn gene. Hum. Mol. Genet. 7: 1417– 1424. Nussbaum, R. L., and Polymeropoulos, M. H. (1997). Genetics of Parkinson’s disease. Hum. Mol. Genet. 6: 1687–1691. Petersen, K., Olesen, O. F., Perregaard, J. K., and Mikkelsen, J. D. (1997). Synuclein/NACP mRNA expression in rat hippocampus and cerebral cortex during development. In “Abstracts of the 27th Annual Meeting of the Society for Neuroscience, New Orleans, LA,” Vol. 23, p. 267. Shibayama-Imazu, T., Okahashi, I., Omata, K., Nakajo, S., Ochiai, H., Nakai, Y., Hama, T., Nakamura, Y., and Nakaya, K. (1993). Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14). Brain Res. 622: 17–25. Trojanowski, J. Q., and Lee, V. M. (1998). Aggregation of neurofilament and a-synuclein proteins in Lewy bodies: Implications for the pathogenesis of Parkinson disease and Lewy body dementia. Arch. Neurol. 55: 151–152. Ue´da, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A. C., Kondo, J., Ihara, Y., and Saitoh, T. (1993). Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. USA 90: 11282–11286.
SHORT COMMUNICATION Genomic Structure and Chromosomal Localization of the Mouse Persyn Gene Maria V. Alimova-Kost,* ,† Natalia N. Ninkina,‡ ,§ Stefan Imreh,* Nikolai V. Gnuchev,‡ Jimi Adu,§ Alun M. Davies,§ ,¶ and Vladimir L. Buchman† ,§ ,1 *Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Box 280, S-17177, Stockholm, Sweden; †Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, 117984, Moscow, Russia; ‡Institute of Gene Biology, Russian Academy of Sciences, 34/6, Vavilov Street, Moscow B-334, Russia; §School of Biomedical Sciences, University of St. Andrews, Bute Medical Buildings, St. Andrews, Fife, KY16 9TS, Scotland, United Kingdom; and ¶Neuropa Ltd., Robertson Building, Dumbarton Road, Glasgow G11 6NU, Scotland, United Kingdom Received July 30, 1998; accepted November 17, 1998
Synucleins are a family of small intracellular proteins expressed mainly in the nervous system. The involvement of synucleins in neurodegeneration and malignancy has been demonstrated, but the physiological functions of these proteins remain elusive. Further studies including generation of animals with modified persyn expression are necessary to clarify the functions of these proteins and the mechanisms of their involvement in human diseases. We cloned and determined the organization and chromosomal localization of the mouse gene coding for persyn, a member of the synuclein family. The gene is composed of five exons, and its general structure is very similar to that of the human persyn gene. Using fluorescence in situ hybridization, we assigned the persyn gene to the boundary of bands B and C on mouse chromosome 14. We found a fragment of the gene that directs expression of the persyn protein in sensory neurons and could be used for generation of transgenic animals. © 1999 Academic Press
Persyn is the most recently characterized member of the synuclein family that also includes a-synuclein/ NACP and b-synuclein/PNP 14 (for review see 1, 5). a-synuclein attracted intent attention after a recent demonstration of its direct involvement in the etiology and pathogenesis of serious human neurodegenerative conditions such as Parkinson and Alzheimer diseases (for review see 1, 4, 5, 8, 15, 18). All three members of the synuclein family are found predominantly in neurons but their spatial distribution in the nervous system and developmental time course of expression are Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. AF017255, AF099984, AF099985, and AF099986. 1 To whom correspondence should be addressed. Telephone: 441334-463282. Fax: 44-1334-463600. E-mail: [email protected].
Genomics 56, 224 –227 (1999) Article ID geno.1998.5674 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
very different. a- and b-synucleins are expressed in the central nervous system, predominantly in the brain (10, 12, 13, 17, 19), but persyn is expressed predominantly in the peripheral nervous system and in motoneurons of the spinal cord and motor ganglia (2, 3). Onsets of a- and b-synuclein expression in brain coincide with the end of the embryonic period of development (9, 17); the levels of expression gradually increase during postnatal development and decrease in aging brains (9, 16, 17). In contrast, persyn expression in peripheral sensory and central motoneurons becomes prominent from the very early stages of their development, gradually increasing during the midgestation period, and is steadily high in adults (2, 3). Persyn expression becomes detectable in the cerebral cortex only in adults and, in contrast to a-synuclein, increases with age (2, 16). In addition, a very high level of persyn expression was detected in malignant breast tumors but not in normal breast tissue or benign breast tumors (11, 14). The mechanisms that regulate the spatial and temporal patterns of expression of synucleins are not known. However, understanding these mechanisms is very important for clarifying the biological functions of these proteins and their involvement in human diseases. Of no less importance will be generation of mice with targeted disruption of synuclein genes. The essential step in this direction is to reveal the organization of mouse genes coding for synucleins. We cloned the persyn gene from a mouse genomic library (Stratagene). Four overlapping l-clones were isolated by hybridization with a mouse persyn cDNA clone (3) and mapped using variety of restriction endonucleases (Fig. 1a). The positions of five exons of the gene were determined by a combination of hybridization and PCR approaches. All exons and adjacent regions of introns were sequenced, and no differences between cDNA and exon sequences were found. The general organization, positions of exon–intron bound-
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225
FIG. 1. (a) Physical map of the mouse persyn genomic locus. (Top) Positions of l clones isolated from a genomic library on the physical map of persyn locus. (Bottom) Detailed map of the mouse persyn gene and a KpnI–BamHI construct used for microinjections and transfections (see text). Positions of exons (black boxes) and the initiation ATG codon (arrow) are shown. (b– d) Localization of persyn gene on mouse metaphase chromosomes using fluorescence in situ hybridization. (b) Metaphase plate; arrows show specific hybridization signals on chromosome 14 obtained with the mixture of biotin-labeled probes lPS11 and lPS91. (c) The same metaphase plate, inverted DAPI banding (black arrows show borders of DAPI bands B and C that hybridized with the probe). (d) Magnified images of chromosome 14 from different metaphase plates. Two images are shown for each chromosome—the FISH signal and inverted DAPI banding.
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aries, and even sizes of introns of the mouse persyn gene are very similar to those of the human persyn gene (14). We used biotin-labeled DNA of clones lPS11 and lPS91 as probes for FISH localization of the persyn gene on mouse metaphase chromosomes. The gene was assigned to the boundary of bands B and C on mouse chromosome 14 (Figs. 1b–1d). This region has syntenic relationships with the 10q23.2– q23.3 region of the human genome where we have localized the human persyn gene (14) as there are a few other known genes (for instance glutamate dehydrogenase gene) localized to these regions of human chromosome 10 and mouse chromosome 14. Information regarding the organization of the mouse persyn gene is very important for further studies of persyn function, particularly for generation of knockout mice. Another powerful approach for studying persyn function is the generation of transgenic mice with increased expression of persyn in cells that normally express this protein. This is especially interesting in light of our recent data about disruption of the neurofilament network in cultured sensory neurons overexpressing persyn (2). The same effect of persyn overexpression in sensory and motoneurons of transgenic animals will suggest the possible involvement of this protein in human neurodegenerative diseases such as giant axonal neuropathy and amyotrophic lateral sclerosis, conditions that severely affect neuronal neurofilaments (6, 7). The best strategy to achieve the right developmental- and cell-specific expression of the protein in transgenic animals is to use the natural regulatory sequences of the gene. To check whether or not a fragment of the mouse persyn gene will direct persyn protein expression in neurons, we constructed a plasmid that contained the whole mouse persyn gene including fragments of upstream and downstream regions but without the internal part (between the NcoI and the XbaI sites) of long intron 3 (see Fig. 1a). This plasmid was either microinjected into cultured mouse sensory neurons or transfected into mouse 3T3 fibroblasts. Forty-eight hours later, cells were fixed and stained with anti-persyn antibody (2, 3). Most of the injected neurons demonstrated intensive staining (Fig. 2a), but no stained fibroblasts were detected, although in parallel cultures transfected with mouse persyn cDNA under Mo-MSV LTR promoter, a substantial percentage of cells expressed persyn protein (Fig. 2b). This result suggests that the region of the mouse persyn gene used in these experiments is sufficient to direct expression of the encoded protein in neurons but not in fibroblasts. Thus, this construct or a similar construct could be used for generation of transgenic mice overexpressing persyn in sensory neurons. In conclusion, we determined the organization and mapped the position of the mouse persyn gene on mouse chromosome 14. These results are important for further functional studies of persyn and other members of the synuclein family.
FIG. 2. (a) Micrograph of mouse sensory (P1 trigeminal ganglia) neurons stained with anti-mouse persyn rabbit polyclonal antibody SK-23 and FITC-labeled secondary antibody. Neurons were microinjected with either a mouse persyn gene plasmid described in the text (above the white line) or a vector plasmid (below the white line) and fixed 48 h later. (b) Micrograph of mouse 3T3 fibroblasts stained with anti-mouse persyn rabbit polyclonal antibody SK-23 and FITClabeled secondary antibody. Fibroblasts were transfected with a plasmid containing mouse persyn cDNA under the control of the Mo-MSV LTR promoter and fixed 48 h later. No stained fibroblasts were seen in parallel cultures transfected with a mouse persyn gene plasmid that directed persyn synthesis in sensory neurons, as illustrated in (a).
ACKNOWLEDGMENTS Our thanks go to Liz Delaney for technical assistance. This work was supported by grants from The Wellcome Trust, The Royal Society, and the Russian Government.
REFERENCES 1.
Buchman, V. L. (1998). Synucleins: Clues to mechanisms of neurodegenerative diseases? Mol. Biol. 32: 592–597. 2. Buchman, V. L., Adu, J., Pino˜n, L. G. P., Ninkina, N. N., and Davies, A. M. (1998). Persyn, a member of the synuclein family, influences neurofilament network integrity. Nature Neurosci. 1: 101–103. 3. Buchman, V. L., Hunter, H. J. A., Pino˜n, L. G. P., Thompson, J., Privalova, E. M., Ninkina, N. N., Lowe, J., and Davies, A. M. (1998). Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system. J. Neurosci. 18: 9335–9341. 4. Chase, T. N. (1997). A gene for Parkinson disease. Arch. Neurol. 54: 1156 –1157.
SHORT COMMUNICATION 5.
6. 7.
8. 9.
10.
11.
12.
13.
Clayton, D. F., and George, J. M. (1998). The synucleins: A family of proteins involved in synaptic function, plasticity, neurodegeneration and disease. Trends Neurosci. 21: 249 –254. Cleveland, D. W. (1996). Neuronal growth and death: Order and disorder in the axoplasm. Cell 84: 663– 666. Donaghy, M., King, R. H., Thomas, P. K., and Workman, J. M. (1988). Abnormalities of the axonal cytoskeleton in giant axonal neuropathy. J. Neurocytol. 17: 197–208. Heintz, N., and Zoghbi, H. (1997). a-synuclein—A link between Parkinson and Alzheimer diseases? Nat. Genet. 16: 325–327. Hsu, L. J., Mallory, M., Xia, Y., Veinbergs, I., Hashimoto, M., Yoshimoto, M., Thal, L. J., Saitoh, T., and Masliah, E. (1998). Expression pattern of synucleins (non-Ab component of Alzheimer’s disease amyloid precursor protein/a-synuclein) during murine brain development. J. Neurochem. 71: 338 – 344. Jakes, R., Spillantini, M. G., and Goedert, M. (1994). Identification of two distinct synucleins from human brain. FEBS Lett. 345: 27–32. Ji, H., Liu, Y. E., Jia, T., Wang, M., Liu, J., Xiao, G., Joseph, B. K., Rosen, C., and Shi, Y. E. (1997). Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing. Cancer Res. 57: 759 –764. Maroteaux, L., and Scheller, R. H. (1991). The rat brain synucleins: Family of proteins transiently associated with neuronal membrane. Mol. Brain Res. 11: 335–343. Nakajo, S., Shioda, S., Nakai, Y., and Nakaya, K. (1994). Localization of phosphoneuroprotein 14 (PNP 14) and its mRNA
14.
15. 16.
17.
18.
19.
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expression in rat brain determined by immunocytochemistry and in situ hybridization. Mol. Brain Res. 27: 81– 86. Ninkina, N., Alimova-Kost, M. V., Paterson, J. W. E., Delaney, L., Cohen, B. B., Imreh, S., Gnuchev, N. V., Davies, A. M., and Buchman, V. L. (1998). Organisation, expression and polymorphism of the human persyn gene. Hum. Mol. Genet. 7: 1417– 1424. Nussbaum, R. L., and Polymeropoulos, M. H. (1997). Genetics of Parkinson’s disease. Hum. Mol. Genet. 6: 1687–1691. Petersen, K., Olesen, O. F., Perregaard, J. K., and Mikkelsen, J. D. (1997). Synuclein/NACP mRNA expression in rat hippocampus and cerebral cortex during development. In “Abstracts of the 27th Annual Meeting of the Society for Neuroscience, New Orleans, LA,” Vol. 23, p. 267. Shibayama-Imazu, T., Okahashi, I., Omata, K., Nakajo, S., Ochiai, H., Nakai, Y., Hama, T., Nakamura, Y., and Nakaya, K. (1993). Cell and tissue distribution and developmental change of neuron specific 14 kDa protein (phosphoneuroprotein 14). Brain Res. 622: 17–25. Trojanowski, J. Q., and Lee, V. M. (1998). Aggregation of neurofilament and a-synuclein proteins in Lewy bodies: Implications for the pathogenesis of Parkinson disease and Lewy body dementia. Arch. Neurol. 55: 151–152. Ue´da, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A. C., Kondo, J., Ihara, Y., and Saitoh, T. (1993). Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. USA 90: 11282–11286.