EXPERIMENTAL
NEUROLOGY
117,97-99
(19%)
BRIEF COMMUNICATION New mRNA Probes for Hippocampal Responses to Entorhinal Cortex Lesions in the Adult Male Rat: A Preliminary Report JONATHAN
R. DAY, BON HONG MIN, NICHOLAS J. LAPING, GLOVER MARTIN HEINZ H. OSTERBURG, AND CALEB E. FINCH
Neurogerontology
Division, Andrus University of Southern
Gerontology California,
Center, and Department Los Angeles, California
of Biological 90089-0191
III,
Sciences,
ECL hippocampus cDNA library was constructed in X ZAP II phage for plaque hybridization screening. To screen for ECL-induced mRNAs, the antisense 32P-labeled ss cDNA probe from ECL and hippocampal mRNA was subtracted using biotinylated sense-strand cDNA (loo-fold excess), which was made from the control phagemid library by Ml3 helper phage rescue. The antisense and sense ss cDNAs were hybridized and the subtracted (or enriched) antisense ss cDNA probes were separated by strepavidin and phenol extraction according to instructions from Invitrogen (San Diego, CA). The subtracted ss cDNA probe and control ds cDNA probes were hybridized with replicate nitrocellulose filters (plaque lifts) from the ECL phage library. This protocol was adapted from the subtractive hybridization method of Duguid et al. (4). From 30,000 plaques, 63 clones were selected on the basis of higher differential autoradiographic plaque intensities compared to the filters hybridized with the control probe. The inserts of the 63 individual phage clones were amplified by polymerase chain reaction using T3 and T7 RNA polymerase primers. Inserts were secondarily screened on slot blots by differential hybridization using cDNA probes from ECL and control hippocampus. Four clones (pECLH-1, pECLH-12, pECLH-29, and pECLH-34; seeTable 1) detected increases in mRNA by Northern blot analysis. Partial sequences were obtained by Sequenase (BRL, Gaithersburg, MD; Fig. 1). Vimentin: pECLH-1 displayed a 50-fold induction in the cortex adjacent to the electrode tract, 4 days after bilateral electrolytic ECL. The clone is 94% similar to mouse vimentin; over the 190 nt sequence displayed, there are 11 mismatches. This cRNA was used to probe for vimentin mRNA in the ipsilateral and contralateral rat hippocampus 2, 4, and 10 days after ECL; a 2.2-kb mRNA species increased twofold in the ipsilateral hippocampus 4 days after unilateral ECL. By in situ hybridization, most of the vimentin mRNA signal was in the ependymal layer of the adjacent ventricles, but not in
Three new mRNA responses were found in the hippocampus of the adult male rat after entorhinal cortex lesions (ECL) that induce synaptic reorganization. Hippocampus cDNA libraries were screened by a subtractive hybridization strategy designed to detect ECL-induced mRNAs. Partial sequencing showed clones with similarities to mouse vimentin, ferritin, and polypeptide 7B-2. A sequence similar to mouse SNAP-25 sequence was also detected. Using a mouse SNAP-25 probe, rat SNAP-25 mRNA increased in the hippocampus after ECL. 0 lew Academic Press, 1~.
We report cDNA clones that detected increased hippocampus mRNA after entorhinal cortex lesions (ECL). These deafferenting lesions induce glial reactivity and synaptic reorganization in the hippocampus (3, 5, 6, 9). These clones may be useful for the study of compensatory mechanisms induced by deafferenting lesions and other types of brain injury. cDNA libraries were constructed with mRNA from the ipsilateral hippocampus of adult male rats (3 months old, F344) 14 days after unilateral electrolytic lesions of the entorhinal cortex (5,9). The 14-day postECL time corresponds to the period of maximum synaptogenesis (3). Poly(A) mRNAs were purified from control or from ipsilateral hippocampi 14 days postECL (pooled, N = 17 each). Directional cloning was based on protocols from the supplier of the vectors (Stratagene, La Jolla, CA); the 5’-end of doublestranded (ds) cDNA was ligated with an EcoRT adaptor and 3’-linked to a XhoI-oligo(dT) adapter primer. The cDNAs from ECL and control hippocampus mRNA had similar sizes (range, 0.5-4.0 kb). The control hippocampus cDNA library was constructed unidirectionally in phagemid pBluescript SK(+) and grown in Escherichia coli XL-l host cells. Ml3 helper phages were used to make sense single-stranded (ss) cDNA in excess. The 97
Copyright 0 1992 All rights of reproduction
0014.4886192 $5.00 by Academic Press, Inc. in any form reserved.
98
BRIEF
COMMUNICATION
TABLE1 ECL-Induced Name
Identity
pECLH-1 pECLH-12 pECLH-29 pECLH-34
Increases
% Similarity
Vimentin SNAP-25 Ferritin 7B2
in Hippocampal
Sequencednt
95 90 90 98
0.19 kb 0.14 kb 0.3 kb 0.11 kb
mRNAs Insert
2.0 kb” 0.4 kb 0.4 kb 0.9 kb
mRNA
2.2 kb 2.0 kb 0.8 kb 1.4 kb
size
Increase Twofold No change’ Twofold Twofold
Note. Insert length was determined by restriction digest of lambda clones and electrophoresis on agarose gels. Percentage similarity refers to the comparison of the sequenced clone to the corresponding segment from mouse in GenBank. Sequence comparisons were made within coding regions. a The vimentin clone was a full-length, coding region clone obtained by rescreening the library with the original 0.4-kb insert. This was done to confirm that full-length clones were present in the library. b Geddes et al. (16) reported an increase in ipsilateral dentate granule neurons after unilateral ECL by in situ hybridization. However, we found no change in mRNA 2 days after ECL by Northern blot hybridization, despite differential hybridization during secondary screening to the 14&y lesion library.
the hippocampal parenchyma. Similar findings were made in the striatum after decortication (G. Pasinetti et aL, unpublished). pEasi-1: mvimen: (1502-1690)
pECLJi-12: mSNAP-25: (1661-1800)
pBCLH-29: mFERRTN: (223-520)
pECLJi-34: a782: (479-590)
TGCACtgAGc TGCACcaAGt
cTGTGCACTT gTGTGCAaTT
TTTATTCAAG TI'TATTCAAG
GTCATCGTGg GTCATCGTGa
TGCTGAGAAG TGCTGAGAAG
TCTCATTGAT TCTCATTGAT
CACCTGTCCg CACCTGTCCa
TCTCTGGTtT TCTCTGGTCT
CAACCGTCTT CAACCGTCTT
AATCAGGAGT AATCAGGAGT
GTTCTTTTgG GTTCTTTTtG
AGTGGGTGTC MCCAGAGGA AGTGGGTGTC AACCAGAGGA
AtGTGACTCC A-GTGACTCC
AGGTTAGTTT AGGTTAGTTT
CTCTCAGGTT CTCTCAGGTT
CAGGAAGAAA CAGGMGMA
GtTl'GGaAGA Gg'ITGGcAGA
GCAGAGAA GCAGAGM
tcgTATGaTT ataTATG_TT
TGGCTGAAAC TGGCTGAAAC
TATTGTAAAT TATTGTAAAT
GGGTGTAATA GGGTGTAATA
TAGGGTTTGT TAGGGTTTGT
CGMTGCTTT CGMTGCWT
TGAAAGCTCT TGAAAGCTCT
GTTTTCCAGA GTTTTCCAGA
CMTACTCTT CAATACTCTT
GTGTGGAAM GTGTGGAAAA
CGTGAAGATC CGTGMGATC
TTCTAAGTCT TTCTAAGTCT
GGCTCTTGtT GGCTCTTG-T
GATCACCAtt GATCACCAaa
GTTgcTgTcg G'ITatTtTqa
CCGGGATGAT CCGGGATGAT
GTGGCCCTGA GTGGCCCTGA
AGMCTTTGC AGMCTTTGC
CAAATACTTT CAAATACTTT
cCTCCATCM -CTCCATCM
TCTCATGAAG TCTCATGMG
AGAGGGAaCA
AGAGGGAgCA
TGCTGAGAAA TGCTGAGAM
CTGATGMGC CTGATGMGC
TGCAGAACCA TGCAGAACCA
GCGAGGTGGA GCGAGGTGGA
CGAATCTTCC CGAATCTTCC
TGCAGGATAT TGCAGGATAT
AAAGAAACCT AAAGAAACCT
GACCGTGATG GACCGTGATG
ACTGGGAGAG ACTGGGAGAG
CGGGCTGCAA CGGGCTGCM
TGGAGTGTGC ACTGCATTGG TGGAGTGTGC ACTGCATTGG
AAAAGAGTGT AAAAGAGTGT
GMTCAgTCA
GMTCAaTCA
CTACTGGMC CTACTGGMC
TTCACAAAcC TTCACAAA-C
TGGCTACTGA TGGCTACTGA
CMGMTGAC
CMGMTGAC
CCACTTATGa CCACTTATGt
GACgatAtTG GACttcAtTG
aAtgaGCAgg -AgacGCA-t
TgaaTcA TaccTgA
CCCCtGACAC CCCCcGACAC
TGCAGAG'ITC TGCAGAGTTC
AGCCGAGMT AGCCGAGMT
TCCAGTTAGA TCCAGTTAGA
CCAGCACCTT CCAGCACCTT
TTTGATCCAG TTTGATCCAC
MCATGACPA AACATGACTA
CCCAGGTTTG CCCAGGTTTG
GGCAAGTGGA GGCAAGTGGA
ACMGAAACG ACMGAAACG
CCTTTATGAG CCTTTATGAG
M M
FIG. 1. Clones isolated from libraries made from hippocampus mRNA after entorhinal cortex lesion. Comparisons of sequence similarities were made using the GenBank database. Capital letters show exact nucleotide identity shared by the rat clone (upper line) and the mouse counterpart (lower line). Lowercase letters show nucleotide mismatches. All sequence comparisons were made within the coding region.
Vimentin is expressed in early development of the rat brain and partially or completely replaced by GFAP and other intermediate filaments beginning in the first neonatal week (17). Vimentin can be induced by plateletderived growth factor (PDGF) (14). Its 5’ regulatory sequence has AP-l/Jun binding sites (15), suggesting that proto-oncogenes and/or growth factors have a role in reactivating fetal genes during synaptic remodeling. In the adult rat brain, vimentin is expressed in capillaries, blood vessels, and tanycytes (16). The striking elevation of vimentin mRNA after ECL implies tanycyte hypertrophy or proliferation during reactive synaptogenesis. SNAP-25: pECLH-12 has strong similarity to synaptosomal-associated protein-25 (SNAP-25) (12). There is a 90% sequence identity to mouse SNAP-25 over the 145 nt that was sequenced, with 7 mismatches. Within this 145 nt was a continuous sequence of 110 nt that contained no mismatches. Thus, pECLH-12 might be a novel SNAP-25 homologue. SNAP-25 may be a presynaptic protein (8). After ECL, in situ hybridization and immunocytochemistry showed increases in the deafferented molecular layer of the dentate gyrus (6). Ferritin: pECLH-29 has 90% similarity to mouse ferritin (2) and increased twofold after ECL. The sequence has 297 nt with 23 mismatches from mouse ferritin which are mostly at the ends of the fragment. This may be the first report of changes in this Fe carrier protein in the hippocampus after brain lesions. Ferritin’s role in responses to neurodegeneration remains undescribed. Ferritin mRNA has an Fe binding stemloop structure that regulates its metabolic stability; a similar structure in amyloid precursor protein mRNA may be disrupted by a mutation (APP717) in a rare familial form of Alzheimer’s disease (18). 7B2: pECLH-34 (twofold increase in hippocampus postECL) is 98% identical to the mouse 7B2 sequence published by Mbikay et al. (11) and 88% similar to human polypeptide 7B2. The 112 nt sequenced showed 1 mismatch to the mouse sequence. 7B2 mRNAs shows
BRIEF
99
COMMUNICATION
1.2- and 1.4-kb bands on Northern blots; these multiple sequences may result from alternate polyadenylation (10,13). Generally, 7B2 is considered a neuroendocrineassociated protein. 7B2 immunoreactivity occurs throughout the brain (7) and also in secretory vesicles of anterior pituitary cells (1). 7B2 was also cloned from a rat insulinoma library (19). The 7B2 polypeptide sequence has a signal peptide sequence and a putative GTP-binding domain; it may have a role in signal transduction (7, 10). These preliminary findings may have value to others who study responses to deafferenting lesions and other models for aspects of Alzheimer’s disease.
1987. Evidence for brain, cerebrospinal controls and patients
protein (7B2) in human Brain concentrations in disease. Peptides 8: 593-
598.
”
LOE~Y, A., W.-S. LIU, C., BAITINGER, AND M. B. WILLARD. 1991. The major %S-methionine-labeled rapidly transported protein (superprotein) is identical to SNAP-25, a protein of synaptic terminals. J. Neurosci. 11: 3412-3421.
9.
LYNCH, G. S., D.A. MATTHEWS, S. MOSKO, T. PARKS, AND C. W. COTMAN. 1972. Induction of acetylcholine esterase rich layer in the dentate gyrus following entorhinal lesions. Brain Res. 42: 311-318.
lo.
MARTENS, J. M. 1988. Cloning and sequence analysis of human pituitary cDNA encoding the novel polypeptide 7B2. Fed. Erp. Biol. Sci. Lett. 234: 160-164.
11.
MBIKAY, M., S. G. N. GRANT, F. SIROIS, H. TADROS, J. SKOWRONSKI, C. LAZURE, N. G. SEIDAH, AND D. HANAHAN. 1989. cDNA sequence of neuroendocrine protein 7B2 expressed in beta cell tumors of transgenic mice. Znt. J. Pept. Protein Res. 33: 39-45.
12.
OYLER, G. A., G. A. HIGGINS, R. A. HART, E. BATTENBERG, F. E. BLOOM, AND M. C. WILSON. 1989. The sequence and characterization of a neuronal specific mRNA encoding a novel synaptosomal associated protein. SNAP-25. J. Cell Biol. 109: 3039-
ACKNOWLEDGMENTS This research was supported by grants from the NIA (AG-7909) and the J.D. and C.T. MacArthur Foundation Program in Successful Aging. N.J.L. was supported by PHS NRSA (AG 05528).
a novel pituitary fluid and plasma: with Alzheimer’s
REFERENCES
3052. 1.
AYOUBI, T. A. Y., H. L. P. VAN DUIJNHOVEN, W. J. M. VAN DE VEN, B. G. JENKS, E. W. ROUBOS, AND G. J. M. MARTENS. 1990. The neuroendocrine polypeptide 7B2 is a precursor protein. J. Biol. Chem. 265: l&644-15,647.
2. BEAUMONT, GRANDCHAMP. and L subunits Unambiguous vitro formation
C., I. DUGAST, F. RENAUDIE, M. SOUROUJON, AND B. 1989. Transcriptional regulation of ferritin H in adult erythroid and liver cells from the mouse: identification of mouse ferritin subunits and in of the ferritin shells. j. Biof. Chem. 264: 749%
7504. 3. CARERES,
A., AND 0. STEWARD. in the denervated dentate gyrus cortex lesions. J. Comp. Neurol.
4. DUGUID,
J. R., R. G. ROHWER, cDNAs of scrapie-modulated tion of a cDNA library. Proc.
1983. Dendritic reorganization of the rat following entorhinal
214: 387-403. AND B. SEED. 1988. Isolation of RNAs by subtractive hybridizaNatl. Acad. Sci. USA 85: 5738-
13.
PAQUET, L., C. LAZURE, N. G. SEIDAH, M. CHRETIEN, AND M. MBIKAY. 1991. The production by alternate splicing of two mRNAs differing by one codon could be an intrinsic property of neuroendocrine protein 7B2 gene expression in man. Biochem. Biophys Res. Commun. 174: 156-162.
14.
RITTLING, S. R., AND R. BASERGA. 1987. Functional analysis growth factor regulation of the human vimentin promoter. Cell. Biol. 7: 3908-3915.
15. RITTLING, AP-l/Jun vimentin
SHAW, G., M. OSBORN, AND K. WEBER. 1981. An immunofluorescence microscopic study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic proein within the adult rat brain. Eur. J. Cell Biol. 26: 68-82.
17
STICHEL, C. C., C. M. MULLER, AND K. ZILLES. 1991. Distribution of glial fibrillary acidic protein and vimentin immunoreactivity during rat visual cortex development. J. Neurocytol. 20: 97-108. TANZI, R. E., AND B. T. HYMAN. 1991. Alzheimer’s mutations. Nature 350: 564.
5. GEDDES, 6.
7. IGUCHI,
H., J. S. D. CHAN,
N. G. SEIDAH,
AND M.
CHRETIEN.
S. R., L. COUTINHO, T. AMRAM, AND M. KOLBE. 1989. binding sites mediate serum inducibility of the human promotor. Nucleic Acids Res. 17: 1619-1633.
16.
5742. J. W., D. T. MONAGHAN, C. W. COTMAN, I. T. LOTT, R. C. KIM, AND H. C. CHUI. 1985. Plasticity of hippocampal circuitry in Alzheimer’s disease. Science 230: 1179-1181. GEDDES, J. W., E. J. HESS, R. A. HART, J. P. KESSLAK, C. W. COTMAN, AND M. C. WILSON. 1990. Lesions of hippocampal circuitry define synaptosomal-associated protein-25 as a novel presynaptic marker. Neurosci. Lett. 109: 54-61.
and Mol.
18 19
WALDBIESER, G. C., J. AIMI, AND J. E. DWON. 1991. Cloning and characterization of the rat complementary deoxyribonucleic acid and gene encoding the neuroendocrine peptide 7B2. Endocrinology 128:3228-3236.
NEUROLOGY
117,97-99
(19%)
BRIEF COMMUNICATION New mRNA Probes for Hippocampal Responses to Entorhinal Cortex Lesions in the Adult Male Rat: A Preliminary Report JONATHAN
R. DAY, BON HONG MIN, NICHOLAS J. LAPING, GLOVER MARTIN HEINZ H. OSTERBURG, AND CALEB E. FINCH
Neurogerontology
Division, Andrus University of Southern
Gerontology California,
Center, and Department Los Angeles, California
of Biological 90089-0191
III,
Sciences,
ECL hippocampus cDNA library was constructed in X ZAP II phage for plaque hybridization screening. To screen for ECL-induced mRNAs, the antisense 32P-labeled ss cDNA probe from ECL and hippocampal mRNA was subtracted using biotinylated sense-strand cDNA (loo-fold excess), which was made from the control phagemid library by Ml3 helper phage rescue. The antisense and sense ss cDNAs were hybridized and the subtracted (or enriched) antisense ss cDNA probes were separated by strepavidin and phenol extraction according to instructions from Invitrogen (San Diego, CA). The subtracted ss cDNA probe and control ds cDNA probes were hybridized with replicate nitrocellulose filters (plaque lifts) from the ECL phage library. This protocol was adapted from the subtractive hybridization method of Duguid et al. (4). From 30,000 plaques, 63 clones were selected on the basis of higher differential autoradiographic plaque intensities compared to the filters hybridized with the control probe. The inserts of the 63 individual phage clones were amplified by polymerase chain reaction using T3 and T7 RNA polymerase primers. Inserts were secondarily screened on slot blots by differential hybridization using cDNA probes from ECL and control hippocampus. Four clones (pECLH-1, pECLH-12, pECLH-29, and pECLH-34; seeTable 1) detected increases in mRNA by Northern blot analysis. Partial sequences were obtained by Sequenase (BRL, Gaithersburg, MD; Fig. 1). Vimentin: pECLH-1 displayed a 50-fold induction in the cortex adjacent to the electrode tract, 4 days after bilateral electrolytic ECL. The clone is 94% similar to mouse vimentin; over the 190 nt sequence displayed, there are 11 mismatches. This cRNA was used to probe for vimentin mRNA in the ipsilateral and contralateral rat hippocampus 2, 4, and 10 days after ECL; a 2.2-kb mRNA species increased twofold in the ipsilateral hippocampus 4 days after unilateral ECL. By in situ hybridization, most of the vimentin mRNA signal was in the ependymal layer of the adjacent ventricles, but not in
Three new mRNA responses were found in the hippocampus of the adult male rat after entorhinal cortex lesions (ECL) that induce synaptic reorganization. Hippocampus cDNA libraries were screened by a subtractive hybridization strategy designed to detect ECL-induced mRNAs. Partial sequencing showed clones with similarities to mouse vimentin, ferritin, and polypeptide 7B-2. A sequence similar to mouse SNAP-25 sequence was also detected. Using a mouse SNAP-25 probe, rat SNAP-25 mRNA increased in the hippocampus after ECL. 0 lew Academic Press, 1~.
We report cDNA clones that detected increased hippocampus mRNA after entorhinal cortex lesions (ECL). These deafferenting lesions induce glial reactivity and synaptic reorganization in the hippocampus (3, 5, 6, 9). These clones may be useful for the study of compensatory mechanisms induced by deafferenting lesions and other types of brain injury. cDNA libraries were constructed with mRNA from the ipsilateral hippocampus of adult male rats (3 months old, F344) 14 days after unilateral electrolytic lesions of the entorhinal cortex (5,9). The 14-day postECL time corresponds to the period of maximum synaptogenesis (3). Poly(A) mRNAs were purified from control or from ipsilateral hippocampi 14 days postECL (pooled, N = 17 each). Directional cloning was based on protocols from the supplier of the vectors (Stratagene, La Jolla, CA); the 5’-end of doublestranded (ds) cDNA was ligated with an EcoRT adaptor and 3’-linked to a XhoI-oligo(dT) adapter primer. The cDNAs from ECL and control hippocampus mRNA had similar sizes (range, 0.5-4.0 kb). The control hippocampus cDNA library was constructed unidirectionally in phagemid pBluescript SK(+) and grown in Escherichia coli XL-l host cells. Ml3 helper phages were used to make sense single-stranded (ss) cDNA in excess. The 97
Copyright 0 1992 All rights of reproduction
0014.4886192 $5.00 by Academic Press, Inc. in any form reserved.
98
BRIEF
COMMUNICATION
TABLE1 ECL-Induced Name
Identity
pECLH-1 pECLH-12 pECLH-29 pECLH-34
Increases
% Similarity
Vimentin SNAP-25 Ferritin 7B2
in Hippocampal
Sequencednt
95 90 90 98
0.19 kb 0.14 kb 0.3 kb 0.11 kb
mRNAs Insert
2.0 kb” 0.4 kb 0.4 kb 0.9 kb
mRNA
2.2 kb 2.0 kb 0.8 kb 1.4 kb
size
Increase Twofold No change’ Twofold Twofold
Note. Insert length was determined by restriction digest of lambda clones and electrophoresis on agarose gels. Percentage similarity refers to the comparison of the sequenced clone to the corresponding segment from mouse in GenBank. Sequence comparisons were made within coding regions. a The vimentin clone was a full-length, coding region clone obtained by rescreening the library with the original 0.4-kb insert. This was done to confirm that full-length clones were present in the library. b Geddes et al. (16) reported an increase in ipsilateral dentate granule neurons after unilateral ECL by in situ hybridization. However, we found no change in mRNA 2 days after ECL by Northern blot hybridization, despite differential hybridization during secondary screening to the 14&y lesion library.
the hippocampal parenchyma. Similar findings were made in the striatum after decortication (G. Pasinetti et aL, unpublished). pEasi-1: mvimen: (1502-1690)
pECLJi-12: mSNAP-25: (1661-1800)
pBCLH-29: mFERRTN: (223-520)
pECLJi-34: a782: (479-590)
TGCACtgAGc TGCACcaAGt
cTGTGCACTT gTGTGCAaTT
TTTATTCAAG TI'TATTCAAG
GTCATCGTGg GTCATCGTGa
TGCTGAGAAG TGCTGAGAAG
TCTCATTGAT TCTCATTGAT
CACCTGTCCg CACCTGTCCa
TCTCTGGTtT TCTCTGGTCT
CAACCGTCTT CAACCGTCTT
AATCAGGAGT AATCAGGAGT
GTTCTTTTgG GTTCTTTTtG
AGTGGGTGTC MCCAGAGGA AGTGGGTGTC AACCAGAGGA
AtGTGACTCC A-GTGACTCC
AGGTTAGTTT AGGTTAGTTT
CTCTCAGGTT CTCTCAGGTT
CAGGAAGAAA CAGGMGMA
GtTl'GGaAGA Gg'ITGGcAGA
GCAGAGAA GCAGAGM
tcgTATGaTT ataTATG_TT
TGGCTGAAAC TGGCTGAAAC
TATTGTAAAT TATTGTAAAT
GGGTGTAATA GGGTGTAATA
TAGGGTTTGT TAGGGTTTGT
CGMTGCTTT CGMTGCWT
TGAAAGCTCT TGAAAGCTCT
GTTTTCCAGA GTTTTCCAGA
CMTACTCTT CAATACTCTT
GTGTGGAAM GTGTGGAAAA
CGTGAAGATC CGTGMGATC
TTCTAAGTCT TTCTAAGTCT
GGCTCTTGtT GGCTCTTG-T
GATCACCAtt GATCACCAaa
GTTgcTgTcg G'ITatTtTqa
CCGGGATGAT CCGGGATGAT
GTGGCCCTGA GTGGCCCTGA
AGMCTTTGC AGMCTTTGC
CAAATACTTT CAAATACTTT
cCTCCATCM -CTCCATCM
TCTCATGAAG TCTCATGMG
AGAGGGAaCA
AGAGGGAgCA
TGCTGAGAAA TGCTGAGAM
CTGATGMGC CTGATGMGC
TGCAGAACCA TGCAGAACCA
GCGAGGTGGA GCGAGGTGGA
CGAATCTTCC CGAATCTTCC
TGCAGGATAT TGCAGGATAT
AAAGAAACCT AAAGAAACCT
GACCGTGATG GACCGTGATG
ACTGGGAGAG ACTGGGAGAG
CGGGCTGCAA CGGGCTGCM
TGGAGTGTGC ACTGCATTGG TGGAGTGTGC ACTGCATTGG
AAAAGAGTGT AAAAGAGTGT
GMTCAgTCA
GMTCAaTCA
CTACTGGMC CTACTGGMC
TTCACAAAcC TTCACAAA-C
TGGCTACTGA TGGCTACTGA
CMGMTGAC
CMGMTGAC
CCACTTATGa CCACTTATGt
GACgatAtTG GACttcAtTG
aAtgaGCAgg -AgacGCA-t
TgaaTcA TaccTgA
CCCCtGACAC CCCCcGACAC
TGCAGAG'ITC TGCAGAGTTC
AGCCGAGMT AGCCGAGMT
TCCAGTTAGA TCCAGTTAGA
CCAGCACCTT CCAGCACCTT
TTTGATCCAG TTTGATCCAC
MCATGACPA AACATGACTA
CCCAGGTTTG CCCAGGTTTG
GGCAAGTGGA GGCAAGTGGA
ACMGAAACG ACMGAAACG
CCTTTATGAG CCTTTATGAG
M M
FIG. 1. Clones isolated from libraries made from hippocampus mRNA after entorhinal cortex lesion. Comparisons of sequence similarities were made using the GenBank database. Capital letters show exact nucleotide identity shared by the rat clone (upper line) and the mouse counterpart (lower line). Lowercase letters show nucleotide mismatches. All sequence comparisons were made within the coding region.
Vimentin is expressed in early development of the rat brain and partially or completely replaced by GFAP and other intermediate filaments beginning in the first neonatal week (17). Vimentin can be induced by plateletderived growth factor (PDGF) (14). Its 5’ regulatory sequence has AP-l/Jun binding sites (15), suggesting that proto-oncogenes and/or growth factors have a role in reactivating fetal genes during synaptic remodeling. In the adult rat brain, vimentin is expressed in capillaries, blood vessels, and tanycytes (16). The striking elevation of vimentin mRNA after ECL implies tanycyte hypertrophy or proliferation during reactive synaptogenesis. SNAP-25: pECLH-12 has strong similarity to synaptosomal-associated protein-25 (SNAP-25) (12). There is a 90% sequence identity to mouse SNAP-25 over the 145 nt that was sequenced, with 7 mismatches. Within this 145 nt was a continuous sequence of 110 nt that contained no mismatches. Thus, pECLH-12 might be a novel SNAP-25 homologue. SNAP-25 may be a presynaptic protein (8). After ECL, in situ hybridization and immunocytochemistry showed increases in the deafferented molecular layer of the dentate gyrus (6). Ferritin: pECLH-29 has 90% similarity to mouse ferritin (2) and increased twofold after ECL. The sequence has 297 nt with 23 mismatches from mouse ferritin which are mostly at the ends of the fragment. This may be the first report of changes in this Fe carrier protein in the hippocampus after brain lesions. Ferritin’s role in responses to neurodegeneration remains undescribed. Ferritin mRNA has an Fe binding stemloop structure that regulates its metabolic stability; a similar structure in amyloid precursor protein mRNA may be disrupted by a mutation (APP717) in a rare familial form of Alzheimer’s disease (18). 7B2: pECLH-34 (twofold increase in hippocampus postECL) is 98% identical to the mouse 7B2 sequence published by Mbikay et al. (11) and 88% similar to human polypeptide 7B2. The 112 nt sequenced showed 1 mismatch to the mouse sequence. 7B2 mRNAs shows
BRIEF
99
COMMUNICATION
1.2- and 1.4-kb bands on Northern blots; these multiple sequences may result from alternate polyadenylation (10,13). Generally, 7B2 is considered a neuroendocrineassociated protein. 7B2 immunoreactivity occurs throughout the brain (7) and also in secretory vesicles of anterior pituitary cells (1). 7B2 was also cloned from a rat insulinoma library (19). The 7B2 polypeptide sequence has a signal peptide sequence and a putative GTP-binding domain; it may have a role in signal transduction (7, 10). These preliminary findings may have value to others who study responses to deafferenting lesions and other models for aspects of Alzheimer’s disease.
1987. Evidence for brain, cerebrospinal controls and patients
protein (7B2) in human Brain concentrations in disease. Peptides 8: 593-
598.
”
LOE~Y, A., W.-S. LIU, C., BAITINGER, AND M. B. WILLARD. 1991. The major %S-methionine-labeled rapidly transported protein (superprotein) is identical to SNAP-25, a protein of synaptic terminals. J. Neurosci. 11: 3412-3421.
9.
LYNCH, G. S., D.A. MATTHEWS, S. MOSKO, T. PARKS, AND C. W. COTMAN. 1972. Induction of acetylcholine esterase rich layer in the dentate gyrus following entorhinal lesions. Brain Res. 42: 311-318.
lo.
MARTENS, J. M. 1988. Cloning and sequence analysis of human pituitary cDNA encoding the novel polypeptide 7B2. Fed. Erp. Biol. Sci. Lett. 234: 160-164.
11.
MBIKAY, M., S. G. N. GRANT, F. SIROIS, H. TADROS, J. SKOWRONSKI, C. LAZURE, N. G. SEIDAH, AND D. HANAHAN. 1989. cDNA sequence of neuroendocrine protein 7B2 expressed in beta cell tumors of transgenic mice. Znt. J. Pept. Protein Res. 33: 39-45.
12.
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ACKNOWLEDGMENTS This research was supported by grants from the NIA (AG-7909) and the J.D. and C.T. MacArthur Foundation Program in Successful Aging. N.J.L. was supported by PHS NRSA (AG 05528).
a novel pituitary fluid and plasma: with Alzheimer’s
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