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• W2068989864 abstract "Mature cerebellar granule cells in culture die by a process that requires new RNA and protein synthesis when deprived of depolarizing concentrations of potassium. We investigated gene expression during the early phase of the cell death program evoked by potassium deprivation. Using a differential gene display technique, we isolated a cDNA that was increased by potassium deprivation. This cDNA was homologous to the 3′ mRNA end of neuronal pentraxin 1 (NP1), a gene encoding a secreted glycoprotein whose expression is restricted to the nervous system. Reverse-Northern and Northern blot analyses confirmed that treatment with low potassium induces overexpression of NP1 mRNA, with a subsequent increase in NP1 protein levels. Time-course studies indicated that overexpression of NP1 protein reaches a maximum after 4 h of exposure to potassium deprivation and 4 h before significant cell death. Incubation of cerebellar granule cells with an antisense oligodeoxyribonucleotide directed against NP1 mRNA reduced low potassium-evoked NP1 protein levels by 60% and attenuated neuronal death by 50%, whereas incubation with the corresponding sense oligodeoxyribonucleotide was ineffective. Furthermore, acute treatment with lithium significantly inhibited both overexpression of NP1 and cell death evoked by low potassium. These results indicate that NP1 is part of the gene expression program of apoptotic cell death activated by nondepolarizing culture conditions in cerebellar granule cells. Mature cerebellar granule cells in culture die by a process that requires new RNA and protein synthesis when deprived of depolarizing concentrations of potassium. We investigated gene expression during the early phase of the cell death program evoked by potassium deprivation. Using a differential gene display technique, we isolated a cDNA that was increased by potassium deprivation. This cDNA was homologous to the 3′ mRNA end of neuronal pentraxin 1 (NP1), a gene encoding a secreted glycoprotein whose expression is restricted to the nervous system. Reverse-Northern and Northern blot analyses confirmed that treatment with low potassium induces overexpression of NP1 mRNA, with a subsequent increase in NP1 protein levels. Time-course studies indicated that overexpression of NP1 protein reaches a maximum after 4 h of exposure to potassium deprivation and 4 h before significant cell death. Incubation of cerebellar granule cells with an antisense oligodeoxyribonucleotide directed against NP1 mRNA reduced low potassium-evoked NP1 protein levels by 60% and attenuated neuronal death by 50%, whereas incubation with the corresponding sense oligodeoxyribonucleotide was ineffective. Furthermore, acute treatment with lithium significantly inhibited both overexpression of NP1 and cell death evoked by low potassium. These results indicate that NP1 is part of the gene expression program of apoptotic cell death activated by nondepolarizing culture conditions in cerebellar granule cells. glyceraldehyde-3-phosphate dehydrogenase neuronal pentraxin 1 oligodeoxyribonucleotide propidium iodide neuronal activity-related pentraxin days in vitro polyvinylidene difluoride polyacrylamide gel electrophoresis polymerase chain reaction In the central nervous system, during normal embryonic development, the number of neurons is adjusted by activation of a built-in gene expression program that kills unnecessary cells in a process described as programmed cell death (1Burek M.J. Oppenheim R.W. Brain Pathol. 1996; 6: 427-446Crossref PubMed Scopus (215) Google Scholar). This process determines the size and shape of the vertebrate nervous system (2Kuan C.Y. Roth K.A. Flavell R.A. Rakic P. Trends Neurosci. 2000; 23: 291-297Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), and the cell death that results from it exhibits the morphological features of apoptosis (3Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6721) Google Scholar). Apoptotic death is characterized by cell shrinkage, nuclear condensation, DNA fragmentation at internucleosomal sites, and degeneration of cell membrane-bound particles that are phagocytized by macrophages without an inflammatory response. Recent evidence indicates that gene expression-dependent apoptosis is also involved in the pathological death of mature neurons observed in various neurodegenerative disorders, such as Alzheimer's disease, or following brain ischemia or traumatic injury of the central nervous system (4Nicotera P. Leist M. Manzo L. Trends Pharmacol. Sci. 1999; 20: 46-51Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 5Leist M. Nicotera P. Exp. Cell Res. 1998; 239: 183-201Crossref PubMed Scopus (262) Google Scholar, 6Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2770) Google Scholar, 7Dragunow M. Faull R.L. Lawlor P. Beilharz E.J. Singleton K. Walker E.B. Mee E. Neuroreport. 1995; 6: 1053-1057Crossref PubMed Scopus (322) Google Scholar, 8Portera-Cailliau C. Hedreen J.C. Price D.L. Koliatsos V.E. J. Neurosci. 1995; 15: 3775-3787Crossref PubMed Google Scholar). Nonetheless, the mechanisms involved in gene expression-dependent apoptotic death of mature neurons are not well characterized. One of the most documented in vitro models to investigate gene expression-dependent apoptosis in mature neuronal cells is death induced by nondepolarizing culture conditions (9D'mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (850) Google Scholar, 10Miller T.M. Johnson E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar, 11Chang J.Y. Wang J.Z. Neurochem. Res. 1997; 22: 43-48Crossref PubMed Scopus (12) Google Scholar). Cerebellar granule cells require depolarizing concentrations of potassium (25–30 mm) to be maintained in culture (12Gallo V. Kingsbury A. Balazs R. Jorgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar). When these cells are mature, reduction of the extracellular concentration of potassium produces a cell death that is morphologically apoptotic. This cell death evoked by low potassium is associated with DNA fragmentation and requires both new RNA and protein synthesis (9D'mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (850) Google Scholar), because addition of protein or RNA synthesis inhibitors within the first 4 h of exposure to potassium deprivation prevents cell death and results in a complete recovery of the damaged DNA (9D'mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (850) Google Scholar,13Galli C. Meucci O. Scorziello A. Werge T.M. Calissano P. Schettini G. J. Neurosci. 1995; 15: 1172-1179Crossref PubMed Google Scholar, 14Nardi N. Avidan G. Daily D. Zilkhafalb R. Barzilai A. J. Neurochem. 1997; 68: 750-759Crossref PubMed Scopus (75) Google Scholar, 15Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar). Likewise, replacement of high concentrations of potassium within 4 h after treatment with low potassium results in no cell loss. However, the activation of the cell death program becomes irreversible in ∼50% of cerebellar granule cells after 6 h of exposure to low potassium (14Nardi N. Avidan G. Daily D. Zilkhafalb R. Barzilai A. J. Neurochem. 1997; 68: 750-759Crossref PubMed Scopus (75) Google Scholar). In neurons, apoptotic death may be mediated by posttranslational mechanisms as well as by “de novo” expression of death genes. However, it has been proposed that the mechanisms involved in low potassium-mediated apoptosis of granule neurons are similar to those operating in neuronal death during development or following blockade of neuronal activity, which require new mRNA and protein synthesis (9D'mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (850) Google Scholar). Consequently, strategies directed to identify genes whose expression is increased before neuronal cells reach the commitment to die may help to identify new targets for neuroprotection. Based on this assumption, we have investigated gene expression during the early phase of the cell death program. Overexpression of several genes has been associated with cerebellar granule cell death induced by low potassium. Thus, glyceraldehyde-3-phosphate dehydrogenase (GAPDH)1 overexpression has been shown to be involved in the initiation of the apoptotic process evoked by low potassium (16Sunaga K. Takahashi H. Chuang D.M. Ishitani R. Neurosci. Lett. 1995; 200: 133-136Crossref PubMed Scopus (90) Google Scholar, 17Ishitani R. Sunaga K. Tanaka M. Aishita H. Chuang D.M. Mol. Pharmacol. 1997; 51: 542-550Crossref PubMed Scopus (71) Google Scholar). Other genes whose overexpression has been found to mediate apoptosis by potassium deprivation include those for the ICE-related protease CPP32 (18Eldadah B.A. Yakovlev A.G. Faden A.I. J. Neurosci. 1997; 17: 6105-6113Crossref PubMed Google Scholar), c-Jun (10Miller T.M. Johnson E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar), Egr-1 (19Catania M.V. Copani A. Calogero A. Ragonese G.I. Condorelli D.F. Nicoletti F. Neuroscience. 1999; 91: 1529-1538Crossref PubMed Scopus (32) Google Scholar), and the transcription factor of cyclin-dependent kinases E2F-1 (20O'Hare M.J. Hou S.T. Morris E.J. Cregan S.P. Xu Q. Slack R.S. Park D.S. J. Biol. Chem. 2000; 275: 25358-25364Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, the identification of other genes of the neuronal death program is necessary to understand the relationship among the different biochemical mechanisms of the apoptotic signal pathway activated by reduction of synaptic activity. Using a differential gene display technique (21Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4694) Google Scholar), we have investigated gene expression evoked by low potassium in cerebellar granule cells. We now report that treatment with low potassium induces the overexpression of neuronal pentraxin 1 (NP1), a gene that was originally identified and isolated as a rat protein that mediates the calcium-dependent uptake of the snake venom toxin, taipoxin (22Schlimgen A.K. Helms J.A. Vogel H. Perin M.S. Neuron. 1995; 14: 519-526Abstract Full Text PDF PubMed Scopus (142) Google Scholar). NP1 encodes a glycoprotein of an apparent molecular mass of ∼50 kDa that is predicted to be secreted and whose expression is restricted to the nervous system (22Schlimgen A.K. Helms J.A. Vogel H. Perin M.S. Neuron. 1995; 14: 519-526Abstract Full Text PDF PubMed Scopus (142) Google Scholar). Our studies demonstrate that antisense oligodeoxyribonucleotides against NP1 inhibit low potassium-induced cell death. Thus, the present results provide evidence of a new function for NP1 and indicate that NP1 is part of the gene program of apoptotic death in cerebellar granule cells kept under nondepolarizing culture conditions. Primary cultures of cerebellar granule neurons were prepared from 7-day postnatal Harlan Sprague-Dawley rat pups (Harlan) as described previously (23Olmos G. DeGregorio-Rocasolano N. Regalado M.P. Gasull T. Boronat M.A. Trullas R. Villarroel A. Lerma J. Garcia-Sevilla J.A. Br. J. Pharmacol. 1999; 127: 1317-1326Crossref PubMed Scopus (154) Google Scholar, 24Viu E. Zapata A. Capdevila J.L. Fossom L.H. Skolnick P. Trullas R. J. Pharmacol. Exp. Ther. 1998; 285: 527-532PubMed Google Scholar). Procedures involving animals and their care were approved by the ethics committee of the University of Barcelona and conducted in conformity with institutional guidelines that are in compliance with national (Generalitat de Catalunya decree 214/1997, DOGC 2450) and international (Guide for the Care and Use of Laboratory Animals, National Institutes of Health publication 85-23, 1985) laws and policies. Cells were dissociated in the presence of trypsin and DNase I, and plated in dishes coated with poly-l-lysine (100 μg/ml). Granule cells were seeded at a density of 3 × 105cells/cm2 in basal Eagle's medium supplemented with 10% fetal bovine serum (heat-inactivated), 0,1 mg/ml gentamicin, 2 mm l-glutamine, and 25 mm KCl. Cerebellar granule cell cultures were kept at 37 °C in a humidified incubator with 5% CO2, 95% air and remained undisturbed until experiments were performed. The replication of non-neuronal cells was prevented by addition of 10 μmcytosine-d-arabinofuranoside to the culture medium 24 h after plating. Cells were used for experiments after 8 days in culture (8DIV). Previous studies have shown that cerebellar granule cells maintained in medium supplemented with 25 mm K+ undergo apoptotic death when switched to 5 mm K+ (9D'mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (850) Google Scholar). In addition, exposure of cerebellar granule cells to fresh serum-containing medium triggers excitotoxicity (25Yan G.M. Ni B.H. Weller M. Wood K.A. Paul S.M. Brain Res. 1994; 656: 43-51Crossref PubMed Scopus (177) Google Scholar). This neurotoxicity does not occur if conditioned medium is used (25Yan G.M. Ni B.H. Weller M. Wood K.A. Paul S.M. Brain Res. 1994; 656: 43-51Crossref PubMed Scopus (177) Google Scholar). Thus, after 8 days in culture, the medium in which cerebellar granule cells had grown, referred to as conditioned medium (Sc+K+), was replaced with one of the following media: fresh unconditioned serum-free medium supplemented with 25 mm potassium (S−K+) or fresh unconditioned serum-free medium containing 5 mmpotassium (S−K−). Immediately after replacement, cells were incubated at 37 °C for different times up to 24 h. Treatments with lithium and antisense oligodeoxyribonucleotides (ODNs) were performed at 8DIV immediately after the replacement of the media described above. LiCl was added to the cultures at a concentration of 5 mm. In pilot experiments, we found that the optimal concentration of ODNs was 10 μm. A 21-base-long phosphorothioated antisense ODN against the NP1 mRNA and its corresponding sense ODN were obtained from Roche Molecular Biochemicals. The sequences were 5′-GCGTGCGGCGCGGCCGGCCAG-3′ for the NP1 antisense ODN (NP1AS) and 5′-C T GGCCGGCCGCGCCGCACGC-3′ for the corresponding sense ODN (NP1S). The phosphorothioated nucleotides are underlined. The NP1 antisense ODN sequence corresponds to nucleotides 4–24, which immediately follow the first initiation codon of the coding sequence of the NP1 cDNA. Cell death was assessed using propidium iodide (PI) staining. PI is excluded by the plasma membrane of viable cells. Injury to the cytoplasmic membrane allows the entry of PI, which, by interacting with nuclear DNA, yields a bright red fluorescence. In time-course experiments, PI fluorescence was measured in 24-well plates using a CytoFluor 2350 scanner (Millipore, Barcelona, Spain) with 530 nm (25-nm band pass) excitation and 645 nm (40-nm band pass) emission filters. The percentage of nonviable cells was measured using a modification of the method described by Rudolph et al. (26Rudolph J.G. Lemasters J.J. Crews F.T. Neurosci. Lett. 1997; 221: 149-152Crossref PubMed Scopus (19) Google Scholar). Base-line fluorescence F0 was measured immediately after treatment with the corresponding medium and addition of PI (30 μm). Subsequent fluorescence readings were obtained at different times after the beginning of treatment. At the end of the experiment, cells were permeabilized with 375 μm digitonin for 10 min at 37 °C to obtain the maximum fluorescence corresponding to 100% of cell death (Fmax). Percentage of cell death was calculated as follows: % cell death = 100 × (Fn − F0)/(Fmax −F0), where Fn is fluorescence at any given time. Cells were kept in the incubator between measurements. In some experiments, percentage of dead cells was measured counting PI stained over total number of cells using simultaneous fluorescence and phase contrast observation in an epifluorescence microscope. In these experiments, cells were incubated with 10 μm PI for 30 min and fixed in 3.7% paraformaldehyde for 20 min at room temperature before addition of a final glycerol protective layer. Gene expression was assessed with the differential display technique as described by Liang and Pardee (21Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4694) Google Scholar) using the RNA image kit from GenHunter. Total RNA from cerebellar granule cells exposed to S−K+ and S−K− for 2 and 4 h was isolated using the Rneasy mini kit (Qiagen) and treated with DNase I (GenHunter, Nashville, TN). First strand cDNA synthesis and33P-radiolabeled differential display PCR were performed using the RNA image kit (GenHunter). The PCR reactions were performed in duplicate for each treatment. The amplified cDNAs were resolved by electrophoresis using a 6% denaturing polyacrylamide gel. After immobilizing the gel on Whatman no. 3MM paper and drying it for 30 min under vacuum at 80 °C, the gel was exposed to X-Omat AR film (Eastman Kodak Co.) overnight. The autoradiogram and the dried gel were oriented with needle punches to be able to locate in the gel the bands identified in the film. After developing the film, the patterns of amplified cDNA bands were compared among treatments. We chose cDNA bands exhibiting a higher intensity in S−K− compared with S−K+, which indicates that low potassium treatment induces the overexpression of a gene represented in the band. The cDNA bands of interest were excised from the gel, reamplified by PCR with the same set of primers and PCR conditions used in the mRNA display but with a higher concentration of dNTPs, and ligated into the pCR-TRAP cloning vector (GenHunter). Ligated plasmids were transformed into GH-competent cells and plated on LB plates containing 20 μg/ml tetracycline. The pCR-TRAP vector includes a tetracycline-dependent positive selection of plasmids with DNA inserts. Only recombinant plasmids confer antibiotic resistance. To verify overexpression of the cDNA fragments identified by differential display, we used two different procedures: reverse Northern dot blot and Northern blot. Bands excised from the gel may contain different cDNA species. To identify the cDNA that is overexpressed in the band, we used the reverse Northern dot blot technique (27Zhang H. Zhang R. Liang P. Nucleic Acids Res. 1996; 24: 2454-2455Crossref PubMed Scopus (95) Google Scholar). This procedure allows the isolation and identification of cDNAs that actually correspond to differentially expressed mRNAs, from bands excised from a differential display gel. For reverse Northern dot blot experiments, tetracycline-resistant colonies were randomly picked from each plate and lysed by boiling in 50 μl of lysis buffer (0.1% Tween 20 in TE buffer, pH 8.0). The cloned cDNA fragments were amplified using primers flanking the cloning site of the vector. Each PCR product was dot-blotted onto duplicate nylon membranes using a microfiltration system. The membranes were UV-cross-linked and probed with total [32P]cDNA. The [32P]cDNA probes were prepared by reverse transcription of 20 μg of total RNA obtained from cerebellar granule cells control (S−K+) and treated with low potassium (S−K−) for 4 h, in the presence of [α-32P]dCTP (3000 Ci/mmol, PerkinElmer Life Sciences). Equal counts (5–10 × 106 cpm) of the cDNA probes from each treatment, (S−K+) and (S−K−), were heat-denatured and used to probe the duplicate blots. Once cDNA overexpression was confirmed by reverse Northern dot blot, we performed Northern blots to verify whether the selected cDNAs represent overexpression of a single mRNA. Northern blot experiments were also performed to study time course of NP1 expression. Total RNA was isolated using TRIzol reagent (Life Technologies, Inc.). Denatured RNA samples (20 μg of total RNA) from cerebellar granule cells controls (Sc+K+ and S−K+) and treated with low potassium (S−K−) were electrophoresed in 1.3% agarose and 0.66 m formaldehyde gels, transferred to nylon membranes (Hybond-XL, Amersham Pharmacia Biotech), and the RNA was fixed to the membranes by baking for 2 h at 80 °C. Hybridization with 32P-labeled probes and washing conditions were performed as described by the membrane manufacturer. Filters were exposed to BioMax films (Amersham Pharmacia Biotech) with intensifying screens for 12–48 h at −80 °C. The NP1 probe used for Northern blot analysis corresponds to nucleotides 5127–5339 of the NP1 cDNA. The NP1 probe was obtained by PCR amplification of a positive clone identified by reverse Northern dot blot. The PCR product was electrophoresed in agarose and purified using the QIAEX II gel extraction kit (Qiagen) and 32P-labeled using Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech). The β-actin probe was obtained by digestion of a pUC19 vector containing a 1.9-kilobase pair human β-actin insert between BamHI sites. Densitometric values of NP1 mRNA bands were obtained using Kodak DS1 computer software and were normalized with the densitometric values of the corresponding β-actin mRNA band. DNA sequencing was performed with Thermo-Sequenase (Amersham Pharmacia Biotech) using an ABI Prism 377 fluorescent sequencing instrument at the Serveis Cientı́fico Tècnics (University of Barcelona, Barcelona, Spain). Data base searches and sequence comparisons were performed using BLAST and BLASTX search servers at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD). After the corresponding treatments, cerebellar granule cells were solubilized in lysis buffer (5 mm Tris-HCl, 150 mm NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 2 μg/ml aprotinin, 1 μg/ml leupeptin, 100 μg/ml phenylmethylsulfonyl fluoride) and briefly sonicated. The homogenate was centrifuged at 15,000 ×g for 20 min at 4 °C. Total protein concentration was determined using the BCA protein assay kit (Pierce). The polypeptides were separated on 10% SDS-polyacrylamide gel electrophoresis and then electroblotted onto PVDF membranes (Millipore, Bedford, MA) according to the manufacturer's protocol. Blots were preincubated with 5% nonfat dry milk in Tris-buffered saline before immunostaining. For specific immunodetection of NP1 protein, mouse anti-rat NP1 monoclonal antibody (Transduction Laboratories, Los Angeles, CA) and rabbit anti-rat polyclonal NP1 antibody (provided by Drs. Carsten Hopf and Paul Worley, Department of Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD), were used at their appropriate concentrations in a solution containing 0.5% nonfat dry milk and 1% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20. Peroxidase-conjugated goat anti-mouse IgG (Transduction Laboratories, Los Angeles, CA) or peroxidase-conjugated mouse anti-rabbit IgG (Sigma) were used as secondary antibodies. In addition to the measurement of the amount of protein before loading, we used a rabbit anti-actin antibody (Sigma) to control for the amount of protein loaded. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech). Quantification of the intensity of the bands on the films was performed with Kodak DS1 computer software. Densitometry values of the bands representing NP1 immunoreactivity were normalized with the values of the corresponding actin bands. Results are expressed as mean ± S.E. of at least three separate experiments. Statistical significance of differences was examined using independent t tests or using one-way analysis of variance when required. Post hoc multiple comparisons were performed using Student-Newman-Keuls tests. Treatment of mature cerebellar granule cells with low potassium resulted in a time-dependent increase in neuronal death, measured with propidium iodide fluorescence (Fig.1). The loss of neurons was ∼60%, 24 h after switching from high to low extracellular concentration of potassium. In comparison, in the same time period, control cultures kept in conditioned medium containing serum and high potassium (Sc+K+) exhibited 13% cell death (Fig. 1). As additional controls, we used cultures in which conditioned medium was replaced with fresh medium supplemented with high (25 mm) potassium, but without serum (S−K+). In these cultures, cell death was 20% after 24 h of treatment. Serum removal did not have a significant effect on cell death until after 12 h of treatment. Moreover, after 24 h of treatment, cell death by serum removal was only 7% compared with undisturbed Sc+K+controls. Treatment with low potassium (5 mm) did not produce any significant cell death until after 8 h of treatment compared with controls. However, 24 h of exposure to low potassium (S−K−) induced 40% cell death, compared with exposure to serum-free medium containing high potassium (S−K+). To identify genes of the death program activated by low potassium before there is significant cell death, we investigated differential gene expression in cells treated with S−K+ and S−K− for 2 and 4 h. We chose these time points because they are well before any significant differences in cell death can be detected between these two treatments (Fig. 1). Moreover, previous studies have shown that cerebellar granule cells can be rescued from low potassium-induced cell death if RNA synthesis inhibitors are added within the first 4 h of treatment but not after (14Nardi N. Avidan G. Daily D. Zilkhafalb R. Barzilai A. J. Neurochem. 1997; 68: 750-759Crossref PubMed Scopus (75) Google Scholar, 15Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar). To identify genes whose expression is induced before neuronal death, we systematically compared mRNA display patterns between cerebellar granule cells exposed to high potassium (S−K+), as controls, and cells treated with low potassium (S−K−). RNA from cultures treated with either S−K+ or S−K− was isolated after 2 and 4 h of treatment and subjected to differential gene display analysis.33P-labeled PCR was performed with 24 different 5′ arbitrary primers combined with each one of three different 3′-anchored primers, for a total of 72 different primer combinations. PCR reactions were performed in duplicate for each treatment group and time point. The analysis of differential gene expression was performed by comparing the band pattern of all these primer combinations for each treatment. We chose only those bands that showed a consistent differential expression, in both duplicates and in the two time points analyzed, between the two treatments. We found 102 bands that in both duplicates exhibited consistent differential expression between high and low potassium treatments at both 2 and 4 h of treatment. Among all of these, 62 bands indicated mRNA overexpression. We chose only those bands that exhibited at least a 4-fold higher densitometric intensity in low over high potassium treatments. We reamplified, subcloned, and sequenced the cDNA species from 12 bands that attained the criterion difference between high and low potassium treatments at both time periods. Finally, we isolated a cDNA band that showed overexpression after both 2 and 4 h of low potassium treatment, named AP21G1 (Fig. 2). To identify the cDNA fragment overexpressed in this band, several clones obtained from the reamplified cDNA from this band were subjected to reverse Northern dot blot (Fig. 3 A). Reverse Northern analysis showed that 4 out of 5 clones (AP21G1–1, -2, -3, and -5) obtained from the AP21G1 band exhibited higher signal when hybridized with cDNA from low potassium treated cultures than when hybridized with cDNA from control cultures (Fig. 3 A). This result indicates that these clones correspond to the gene whose overexpression was induced by treatment with low potassium and, therefore, the gene that confers the high intensity signal to the AP21G1 band (Fig. 2). PCR amplification of the inserts of the positive clones revealed a fragment of ∼200 base pairs in the four cDNAs. The sequence of the cDNA inserts of the four positive clones was identical, confirming that reverse Northern actually identified a single cDNA that was overexpressed in the AP21G1 band. The sequence of this cDNA overexpressed by low potassium treatment is 213 base pairs long and it is represented in Fig. 3 B. A search of the GenBank™ data base at the National Center for Biotechnology Information using the BLAST program revealed that the AP21G1 fragment is 100% homologous with the 3′-untranslated region of rat NP1 mRNA (GenBank™ accession no. U18772) (22Schlimgen A.K. Helms J.A. Vogel H. Perin M.S. Neuron. 1995; 14: 519-526Abstract Full Text PDF PubMed Scopus (142) Google Scholar).Figure 3Reverse Northern dot blot of clones from AP21G1 band. A, the cDNA from the band AP21G1 represented in Fig. 2 (lane 8) was excised, reamplified, and ligated into the pCR-TRAP cloning vector. The PCR products from five randomly picked colonies (AP21G1–1, -2, -3, -4, and -5) were blotted onto duplicate filters. One of the filters (S−K+) was hybridized with32P-labeled cDNA from cerebellar granule cell cultures control. The other filter was hybridized with 32P-labeled cDNA from cerebellar granule cells treated with low potassium for 4 h (S−K−). B, nucleotide sequence of AP21G1–1 clone cDNA fragment. The cDNA inserts of the AP21G1–1, -2, -3, and -5 clones showing differential expression were sequenced. The nucleotide sequence of the four cDNA inserts was identical. The sequence of the primers used in differential gene display analysis are underlined (H =HindIII site at the 5′ end of the primers).View Large Image Figure ViewerDownlo" @default.
• W2068989864 created "2016-06-24" @default.
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• W2068989864 date "2001-01-01" @default.
• W2068989864 modified "2023-10-17" @default.
• W2068989864 title "Overexpression of Neuronal Pentraxin 1 Is Involved in Neuronal Death Evoked by Low K+ in Cerebellar Granule Cells" @default.
• W2068989864 cites W104870287 @default.
• W2068989864 cites W1499591683 @default.
• W2068989864 cites W1550247880 @default.
• W2068989864 cites W1562796888 @default.
• W2068989864 cites W1589415602 @default.
• W2068989864 cites W1613986623 @default.
• W2068989864 cites W1887007239 @default.
• W2068989864 cites W1966492450 @default.
• W2068989864 cites W1972149163 @default.
• W2068989864 cites W1973198505 @default.
• W2068989864 cites W1973891315 @default.
• W2068989864 cites W1976441097 @default.
• W2068989864 cites W1978368163 @default.
• W2068989864 cites W1979176029 @default.
• W2068989864 cites W1981527579 @default.
• W2068989864 cites W1982768284 @default.
• W2068989864 cites W1989831685 @default.
• W2068989864 cites W1997492755 @default.
• W2068989864 cites W2020181864 @default.
• W2068989864 cites W2024709318 @default.
• W2068989864 cites W2029247738 @default.
• W2068989864 cites W2038027765 @default.
• W2068989864 cites W2046807462 @default.
• W2068989864 cites W2048121649 @default.
• W2068989864 cites W2050517000 @default.
• W2068989864 cites W2064751454 @default.
• W2068989864 cites W2066149485 @default.
• W2068989864 cites W2072286244 @default.
• W2068989864 cites W2073264699 @default.
• W2068989864 cites W2075717402 @default.
• W2068989864 cites W2086153799 @default.
• W2068989864 cites W2092061953 @default.
• W2068989864 cites W2102527735 @default.
• W2068989864 cites W2107100596 @default.
• W2068989864 cites W2109658405 @default.
• W2068989864 cites W2125779862 @default.
• W2068989864 cites W2139488506 @default.
• W2068989864 cites W2169161439 @default.
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